CX3CR1-binding polypeptides comprising immunoglobulin single variable domains

ABSTRACT

The present invention relates to CX3CR1-binding polypeptides, in particular polypeptides comprising specific immunoglobulin domains. The invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions comprising such polypeptides; and to uses of such polypeptides or such compositions, in particular for prophylactic, therapeutic and diagnostic purposes.

FIELD OF THE INVENTION

The present invention relates to CX3CR1-binding polypeptides, inparticular polypeptides comprising specific immunoglobulin domains. Theinvention also relates to nucleic acids encoding such polypeptides; tomethods for preparing such polypeptides; to host cells expressing orcapable of expressing such polypeptides; to compositions comprising suchpolypeptides; and to uses of such polypeptides or such compositions, inparticular for prophylactic, therapeutic and diagnostic purposes.

BACKGROUND OF THE INVENTION

CX3CR1 is a G-protein coupled integral membrane protein, which is achemokine receptor. It is predominantly expressed on cell types such asmonocytes, dendritic cells and T cells that have been associated withthe initiation and progression of atherosclerotic plaques. It isupregulated on monocytes by oxidized lipids and mediates migration ofthese cells into and survival within plaques. Its unique ligandfractalkine (FKN) is expressed on the surface of vascular endothelialand smooth muscle cells in lesions where it modulates leukocyteadhesion. Fractalkine is also released into the circulation byproteolytic cleavage where it functions as a chemotactic agent.

In humans, a CX3CR1 variant (V249I/T280M) with decreased activity hasbeen shown to be associated with a lower risk of cardiovascular disease(coronary heart disease, cerebrovascular disease or peripheral vasculardisease)(McDermott, 2001; Circ Res 89:401), coronary artery disease(angiographic evidence of stenosis) (McDermott, 2003; J. Clin. Invest.111:1241), and carotid artery occlusive disease (Ghilardi, 2004; Stroke35:1276). CX3CR1 co-localized with fractalkine which showed enhancedimmunostaining by polyclonal antibodies within atherosclerotic plaques(Wong, 2002 Cardiovasc. Path. 11:332). No fractalkine staining wasobserved in non-plaque arterial regions.

Several independent mouse genetic studies have shown a beneficial effectof CX3CR1 deficiency on atherosclerosis. A reduction in lesion area inthe aortic arch and thoracic aorta as well as a decrease inmonocyte/macrophage accumulation in plaques was seen in twoindependently derived strains of CX3CR1^(−/−) apoE^(−/−) mice fed a highfat diet (Combadière, 2003; Circulation, 107:1009, Lesnik, 2003; J.Clin. Invest. 111:333).

This shows that CX3CR1 is involved in cardiovascular diseases and themodulation of its activity could provide promising therapies. There istherefore a need for antagonist molecules against CX3CR1 with beneficialpharmacological properties, which can be used as therapeutic agents totreat diseases, in particular cardiovascular diseases in humans.

Accordingly, one aim of the present invention is to provide anti-CX3CR1antagonist molecules, in particular anti-CX3CR1 antagonist molecules,which have high binding affinity to CX3CR1.

A further aim of the present invention is to provide anti-CX3CR1antagonist molecules, which have high specificity for CX3CR1.

A further aim of the present invention is to provide anti-CX3CR1antagonists, which have potent activity.

A further aim of the present invention is to provide anti-CX3CR1antagonists, which have a favorable bioavailability and half-life.

A further aim of the present invention is to provide anti-CX3CR1antagonists, which have favorable biophysical properties.

Further aims of the present invention include combinations of any of theaims set forth above.

SUMMARY OF THE INVENTION

The invention provides polypeptides which bind to human CX3CR1 and arecapable of blocking the binding of human fractalkine to human CX3CR1. Inone aspect, the polypeptide is an immunoglobulin comprising anantigen-binding domain comprising three complementarity determiningregions CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to humanCX3CR1 and is capable of blocking the binding of human fractalkine tohuman CX3CR1. In a further aspect, the polypeptide comprises one or moreanti-CX3CR1 immunoglobulin single variable domain, wherein saidpolypeptide is capable of blocking the binding of human fractalkine tohuman CX3CR1.

In one aspect, a polypeptide of the present invention is characterizedby one or more of the following properties:

-   -   Bind with high affinity to human CX3CR1;    -   Block the binding of soluble fractalkine to human CX3CR1;    -   Inhibit fractalkine induced chemotaxis;    -   Inhibit fractalkine induced human CX3CR1 receptor        internalization;    -   Cross-react with cyno CX3CR1 within 10-fold of E/IC₅₀ for human        CX3CR1 for binding and functional inhibition.

In a further aspect, a polypeptide of the present invention comprises ananti-CX3CR1 immunoglobulin single variable domain and further comprisesa half-life extending moiety, for example an albumin binding moiety, apolyethylene glycol molecule or a Fc domain. In a further aspect, apolypeptide of the present invention comprises two or more anti-CX3CR1immunoglobulin single variable domains. In one aspect, the twoanti-CX3CR1 immunoglobulin single variable domains are covalently linkedby a linker peptide. In one aspect, the two anti-CX3CR1 immunoglobulinsingle variable domains in a polypeptide of the present invention havethe same amino acid sequence. In another aspect, the two anti-CX3CR1immunoglobulin single variable domains in a polypeptide of the presentinvention have different amino acid sequences. In one aspect, apolypeptide of the present invention comprises two anti-CX3CR1immunoglobulin single variable domains and further comprises a half-lifeextending moiety, for example an albumin binding moiety, a polyethyleneglycol molecule or a Fc domain.

In one aspect, a polypeptide of the present invention comprises a firstanti-CX3CR1 immunoglobulin single variable domain covalently linked toan albumin binding moiety by a first linker peptide, wherein saidalbumin binding moiety is further covalently linked to a secondanti-CX3CR1 immunoglobulin single variable domain by a second linkerpeptide.

In one aspect, a polypeptide of the present invention comprises ananti-CX3CR1 immunoglobulin single variable domain covalently linked to aFc domain by a linker peptide. In one aspect, such polypeptidecomprising an anti-CX3CR1 immunoglobulin single variable domaincovalently linked to a Fc domain by a linker peptide is provided as adimer, for example through disulfide bridges. The polypeptides of thepresent invention are used for the prevention, treatment, alleviationand/or diagnosis of CX3CR1-associated diseases, disorders or conditions,in particular cardiovascular diseases, such as atherosclerosis.

In a further aspect, the present invention provides:

Embodiment 1

An immunoglobulin comprising an antigen-binding domain comprising threecomplementarity determining regions CDR1, CDR2 and CDR3, wherein saidimmunoglobulin binds to human CX3CR1 and is capable of blocking thebinding of human fractalkine to human CX3CR1.

Embodiment 2

A polypeptide comprising one or more anti-CX3CR1 immunoglobulin singlevariable domain, wherein said polypeptide is capable of blocking thebinding of human fractalkine to human CX3CR1.

Embodiment 3

A polypeptide according to embodiment 2, wherein said anti-CX3CR1immunoglobulin single variable domain consists essentially of fourframework regions (FR1, FR2, FR3 and FR4) and three complementarydetermining regions (CDR1, CDR2 and CDR3).

Embodiment 4

A polypeptide according to embodiment 3, wherein said anti-CX3CR1immunoglobulin single variable domain has the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Embodiment 5

A polypeptide according to any one of embodiments 2 to 4, wherein saidanti-CX3CR1 immunoglobulin single variable domain is an antibody domain.

Embodiment 6

A polypeptide according to embodiment 5, wherein said anti-CX3CR1immunoglobulin single variable domain is a VH, VL, VHH, camelized VH, orVHH that is optimized for stability, potency, manufacturability and/orsimilarity to human framework regions.

Embodiment 7

A polypeptide according to any one of embodiments 1 to 6, wherein saidpolypeptide has an affinity to human CX3CR1 at:

-   -   a) an EC50 of less than or equal to 10 nM, less than or equal to        5 nM, less than or equal to 2.5 nM or less than or equal to 1        nM, as determined by cell binding FACS; or    -   b) an 1050 of less than or equal to 10 nM, less than or equal to        5 nM, less than or equal to 2.5 nM or less than or equal to 1        nM, as determined by competition FACS.

Embodiment 8

A polypeptide according to any one of embodiments 1 to 7, wherein saidpolypeptide blocks the binding of human fractalkine to human CX3CR1 atan IC₅₀ of less than or equal to 300 nM, or less than or equal to 100nM, or less than or equal to 20 nM, or less than or equal to 10 nM, orless than or equal to 5 nM, or less than or equal to 2.5 nM or less thanor equal to 1 nM.

Embodiment 9

A polypeptide according to any one of embodiments 1 to 8, wherein saidpolypeptide inhibits fractalkine induced chemotaxis mediated by humanCX3CR1 at an IC₅₀ of less than or equal to 500 nM, or of less than orequal to 100 nM, or of less than or equal to 75 nM, or of less than orequal to 50 nM, or less than or equal to 10 nM or less than or equal to5 nM.

Embodiment 10

A polypeptide according to any one of embodiments 1 to 9, wherein saidpolypeptide inhibits fractalkine internalization mediated by humanCX3CR1 at an IC₅₀ of less than or equal to 10 nM, or less than or equalto 5 nM or or less than or equal to 1 nM.

Embodiment 11

A polypeptide according to any one of embodiments 3 to 10, wherein saidCDR3 has the amino acid sequence ofAsp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 197), wherein:

-   -   Xaa1 is Pro, Ala or Gly;    -   Xaa2 is Asp or Asn;    -   Xaa3 is Thr or Ser;    -   Xaa4 is Arg, Lys, Ala or Gly; and    -   Xaa5 is Tyr or Phe.

Embodiment 12

A polypeptide according to any one of embodiments 3 to 11, wherein:

a)

-   -   Xaa1 is Pro, Ala or Gly;    -   Xaa2 is Asp or Asn;    -   Xaa3 is Thr;    -   Xaa4 is Arg or Lys; and    -   Xaa5 is Tyr,

and/or

b) wherein said CDR3 is selected from any of SEQ ID No's: 186-190.

Embodiment 13

A polypeptide according to any one of embodiments 3 to 12, wherein saidCDR3 has the amino acid sequence ofAsp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyr (SEQ ID NO: 186).

Embodiment 14

A polypeptide according to any one of embodiments 3 to 10, wherein:

-   -   i) said CDR1:        -   a) has the amino acid sequence of SEQ ID NO: 141;        -   b) has an amino acid sequence that has at least 80% amino            acid identity with the amino acid sequence of SEQ ID NO:            141;        -   c) has an amino acid sequence that has 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 141,            wherein            -   at position 2 the S has been changed into T, or G;            -   at position 6 the S has been changed into R;            -   at position 7 the N has been changed into T; and/or            -   at position 9 the M has been changed into K; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 141-145 and 213;    -   ii) said CDR2:        -   a) has the amino acid sequence of SEQ ID NO: 164;        -   b) has an amino acid sequence that has at least 70% amino            acid identity with the amino acid sequence of SEQ ID NO:            164;        -   c) has an amino acid sequence that has 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 164, wherein            -   at position 1 the G has been changed into A, L, V or S;            -   at position 3 the N has been changed into D, S, Q, G or                T;            -   at position 4 the S has been changed into T, K, G or P;            -   at position 5 the V has been changed into A;            -   at position 6 the G has been changed into D;            -   at position 7 the I has been changed into T, or V;            -   at position 8 the T has been changed into A; and/or            -   at position 9 the K has been changed into R; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 162-175 and 214-221; and    -   iii) said CDR3:        -   a) has the amino acid sequence of SEQ ID NO: 186;        -   b) has an amino acid sequence that has at least 70% amino            acid identity the amino acid sequence of SEQ ID NO: 186;        -   c) has an amino acid sequence that has 3, 2, or 1 amino            acid(s) difference with the amino acid sequences of SEQ ID            NO: 186, wherein            -   at position 2 the P has been changed into A, or G;            -   at position 7 the D has been changed into N; and/or            -   at position 9 the R has been changed into K; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 186-190.

Embodiment 15

A polypeptide according to any one of embodiments 3 to 10, wherein

-   -   i) said CDR1 has the amino acid sequence of SEQ ID NO: 146;    -   ii) said CDR2 has an amino acid sequence that a) has at least        90% amino acid identity with the amino acid sequence of SEQ ID        NO: 176 orb) has the amino acid sequence of SEQ ID NO: 176 or        177; and    -   iii) said CDR3 has the amino acid sequence of SEQ ID NO: 191.

Embodiment 16

A polypeptide according to any one of embodiments 3 to 10, wherein

-   -   i) said CDR1:        -   a) has the amino acid sequence of SEQ ID NO: 147; or        -   b) has an amino acid sequence that has 6, 5, 4, 3, 2, or 1            amino acid(s) difference with the amino acid sequence of SEQ            ID NO: 147, wherein            -   at position 1 the G has been changed into K, R, or A;            -   at position 2 the T has been changed into I, P, S or L;            -   at position 3 the I has been changed into V, or T;            -   at position 4 the F has been changed into L;            -   at position 5 the S has been changed into R, or D;            -   at position 6 the N has been changed into S, T, or D;                and/or            -   at position 7 the N has been changed into T, or Y; or        -   c) has an amino acid sequence selected from any one of SEQ            ID NO's: 147-161;    -   ii) said CDR2:        -   a) has the amino acid sequence of SEQ ID NO: 179; or        -   b) has an amino acid sequences that has 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 179, wherein            -   at position 3 the S has been changed into T, or G;            -   at position 4 the N has been changed into S, or I;            -   at position 5 the S has been changed into T;            -   at position 6 the G has been changed into Y; and/or            -   at position 8 the T has been changed into A; or        -   c) has an amino acid sequence selected from any one of SEQ            ID NO's: 178-185; and    -   iii) said CDR3:        -   a) has the amino acid sequence of SEQ ID NO: 192; or        -   b) has an amino acid sequence that has at least 80% amino            acid identity with the amino acid sequence of SEQ ID NO:            192; or        -   c) has an amino acid sequence that has 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 192,            wherein            -   at position 2 the A has been changed into G;            -   at position 8 the T has been changed into S;            -   at position 9 the A has been changed into G; and/or            -   at position 10 the Y has been changed into F; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 192-196.

Embodiment 17

A polypeptide according to embodiment 3, wherein the amino acidsequences of said CDR1, CDR2 and CDR3 are set forth in:

-   -   SEQ ID No: 141, 162 and 186, respectively; or    -   SEQ ID No: 141, 163 and 187, respectively; or    -   SEQ ID No: 141, 164 and 186, respectively; or    -   SEQ ID No: 141, 166 and 186, respectively; or    -   SEQ ID No: 141, 167 and 186, respectively; or    -   SEQ ID No: 141, 167 and 189, respectively; or    -   SEQ ID No: 141, 168 and 186, respectively; or    -   SEQ ID No: 141, 168 and 187, respectively; or    -   SEQ ID No: 141, 169 and 190, respectively; or    -   SEQ ID No: 141, 170 and 186, respectively; or    -   SEQ ID No: 141, 171 and 186, respectively; or    -   SEQ ID No: 141, 174 and 186, respectively; or    -   SEQ ID No: 141, 175 and 187, respectively; or    -   SEQ ID No: 142, 165 and 188, respectively; or    -   SEQ ID No: 142, 173 and 188, respectively; or    -   SEQ ID No: 143, 164 and 186, respectively; or    -   SEQ ID No: 144, 172 and 187, respectively; or    -   SEQ ID No: 145, 172 and 187, respectively; or    -   SEQ ID No: 141, 214 and 186, respectively; or    -   SEQ ID No: 141, 215 and 186, respectively; or    -   SEQ ID No: 141, 216 and 186, respectively; or    -   SEQ ID No: 141, 217 and 186, respectively; or    -   SEQ ID No: 141, 218 and 186, respectively; or    -   SEQ ID No: 141, 219 and 186, respectively; or    -   SEQ ID No: 141, 220 and 186, respectively; or    -   SEQ ID No: 213, 221 and 186, respectively; or    -   SEQ ID No: 213, 214 and 186, respectively.

Embodiment 18

A polypeptide according to embodiment 3, wherein the amino acidsequences of said CDR1, CDR2 and CDR3 are set forth in:

-   -   SEQ ID No: 146, 176 and 191, respectively; or    -   SEQ ID No: 146, 177 and 191, respectively.

Embodiment 19

A polypeptide according to embodiment 3, wherein the amino acidsequences of said CDR1, CDR2 and CDR3 are set forth in:

-   -   SEQ ID No: 147, 178 and 192, respectively; or    -   SEQ ID No: 147, 179 and 192, respectively; or    -   SEQ ID No: 147, 179 and 194, respectively; or    -   SEQ ID No: 148, 179 and 193, respectively; or    -   SEQ ID No: 149, 179 and 192, respectively; or    -   SEQ ID No: 149, 180 and 192, respectively; or    -   SEQ ID No: 149, 181 and 192, respectively; or    -   SEQ ID No: 149, 183 and 192, respectively; or    -   SEQ ID No: 149, 185 and 192, respectively; or    -   SEQ ID No: 150, 179 and 194, respectively; or    -   SEQ ID No: 150, 182 and 194, respectively; or    -   SEQ ID No: 151, 179 and 193, respectively; or    -   SEQ ID No: 151, 182 and 194, respectively; or    -   SEQ ID No: 151, 184 and 196, respectively; or    -   SEQ ID No: 152, 179 and 195, respectively; or    -   SEQ ID No: 153, 179 and 194, respectively; or    -   SEQ ID No: 154, 182 and 194, respectively; or    -   SEQ ID No: 155, 179 and 195, respectively; or    -   SEQ ID No: 156, 181 and 192, respectively; or    -   SEQ ID No: 157, 179 and 194, respectively; or    -   SEQ ID No: 158, 179 and 192, respectively; or    -   SEQ ID No: 159, 178 and 192, respectively; or    -   SEQ ID No: 160, 179 and 194, respectively; or    -   SEQ ID No: 161, 179 and 194, respectively.

Embodiment 20

A polypeptide according to embodiment 3, wherein the amino acidsequences of said CDR1, CDR2 and CDR3 are set forth in: SEQ ID NO's:141, 164 and 186, respectively, or SEQ ID NO's: 141, 162 and 186,respectively.

Embodiment 21

A polypeptide according to embodiment 3, wherein the amino acidsequences of said CDR1, CDR2 and CDR3 are set forth in: SEQ ID NO's:213, 214 and 186 respectively, SEQ ID NO's: 213, 221 and 186respectively, or SEQ ID NO's: 141, 162 and 186 respectively.

Embodiment 22

A polypeptide according to any one of embodiments 2 to 10, wherein saidanti-CX3CR1 immunoglobulin single variable domain is a VHH domaincomprising the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 3;    -   b) amino acid sequences that have at least 90% amino acid        identity with the amino acid sequences of SEQ ID NO: 3;    -   c) amino acid sequences that have 11, 10, 9, 8, 7, 6, 5, 4, 3,        2, or 1 amino acid difference with the amino acid sequences of        SEQ ID NO: 3; or    -   d) an amino acid sequence of any one of SEQ ID NO: 1-48, 121-140        or 222-224.

Embodiment 23

A polypeptide according to any one of embodiments 2 to 10, wherein saidanti-CX3CR1 immunoglobulin single variable domain is a VHH domaincomprising the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 49;    -   b) an amino acid sequence that has at least 95% amino acid        identity with the amino acid sequences of SEQ ID NO: 49;    -   c) an amino acid sequence that has 5, 4, 3, 2, or 1 amino acid        difference with the amino acid sequences of SEQ ID NO: 49; or    -   d) an amino acid sequence of any one of SEQ ID NO: 49-52.

Embodiment 24

A polypeptide according to any one of embodiments 2 to 10, wherein saidanti-CX3CR1 immunoglobulin single variable domain is a VHH domaincomprising the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 67;    -   b) an amino acid sequence that has at least 90% amino acid        identity with the amino acid sequences of SEQ ID NO: 67;    -   c) an amino acid sequence that has 12, 11, 10, 9, 8, 7, 6, 5, 4,        3, 2, or 1 amino acid difference with the amino acid sequences        of SEQ ID NO: 67; or    -   d) an amino acid sequence of any one of SEQ ID NO: 53-120.

Embodiment 25

A polypeptide according to embodiment 2, wherein said anti-CX3CR1immunoglobulin single variable domain comprises the sequence set forthin SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment 26

A polypeptide according to embodiment 2, wherein said anti-CX3CR1immunoglobulin single variable domain comprises the sequence set forthin any one of SEQ ID NO: 121-140 or SEQ ID NO: 222-224.

Embodiment 27

A polypeptide according to any of one of the embodiments above, which ishumanized and/or optimized for stability, potency, manufacturabilityand/or similarity to human framework regions.

Embodiment 28

A polypeptide according to embodiment 27, which is humanized and/orsequence optimized in one or more of the following positions (accordingto Kabat numbering): 1, 11, 14, 16, 74, 83, 108.

Embodiment 29

A polypeptide according to embodiment 28, comprising one or more of thefollowing mutations: E1D, S11L, A14P, E16G, A74S, K83R, Q108L.

Embodiment 30

A polypeptide according to any one of embodiments 3-29, in which:

-   -   i) FR1 is selected from SEQ ID NO's: 198-204;    -   ii) FR2 is selected from SEQ ID NO's: 205-208;    -   iii) FR3 is selected form SEQ ID NO's: 209-210; and/or    -   iv) FR4 is selected from SEQ ID NO's: 211-212.

Embodiment 31

A polypeptide according to any one of embodiments 3-30, which ishumanized and/or sequence optimized in one or more of the followingpositions (according to Kabat numbering): 52, 53.

Embodiment 32

A polypeptide according to embodiment 31, comprising one or more of thefollowing mutations: N52S, S53T.

Embodiment 33

A polypeptide according to any one of embodiments 3-32, in which CDR2 isselected from SEQ ID NO's: 214-221.

Embodiment 34

A polypeptide according to any one of embodiments 2-33, wherein saidanti-CX3CR1 immunoglobulin single variable domain comprises the sequenceset forth in any of SEQ ID NO's: 138-140 or 222-224.

Embodiment 35

A polypetide according to any one of embodiments 22 to 26, wherein saidVHH domain consists of any one of said amino acid sequences.

Embodiment 36

A polypeptide according to any one of embodiments 2 to 35, wherein saidimmunoglobulin single variable domain cross-blocks the binding of atleast one of the immunoglobulin single variable domains of SEQ ID NO's:1-120, 121-140 and 222-224 to CX3CR1.

Embodiment 37

A polypeptide according to any one of embodiments 2 to 35, wherein saidimmunoglobulin single variable domain is cross-blocked from binding toCX3CR1 by at least one of the amino acid sequences of SEQ ID NO's:1-120, 121-140 and 222-224.

Embodiment 38

A polypeptide according to any one of embodiments 2 to 37, wherein thepolypeptide further comprises a half-life extending moiety.

Embodiment 39

A polypeptide according to embodiment 38, wherein said half-lifeextending moiety is covalently linked to said polypeptide and isselected from the group consisting of an albumin binding moiety, such asan anti-albumin immunoglobulin domain, a transferrin binding moiety,such as an anti-transferrin immunoglobulin domain, a polyethylene glycolmolecule, a recombinant polyethylene glycol molecule, human serumalbumin, a fragment of human serum albumin, an albumin binding peptideor a Fc domain.

Embodiment 40

A polypeptide according to embodiment 38 or 39, wherein said half-lifeextending moiety consists of an anti-albumin immunoglobulin singlevariable domain.

Embodiment 41

A polypeptide according to embodiment 40, wherein the immunoglobulinsingle variable domain is selected from a VHH domain, a humanized VHHdomain, a camelized VH domain, a domain antibody, a single domainantibody and/or “dAb”s.

Embodiment 42

A polypeptide according to embodiment 41, wherein the anti-albuminimmunoglobulin single variable domain is selected from SEQ ID NO's:230-232.

Embodiment 43

A polypeptide according to any one of embodiment 2 to 39, wherein saidpolypeptide is linked to an Fc portion (such as a human Fc, for exampleas set forth in SEQ ID NO: 252), optionally via a suitable linker orhinge region.

Embodiment 44

A polypeptide according to any one of embodiments 2 to 39, wherein saidpolypeptide is further linked to one or more constant domains (forexample, 2 or 3 constant domains that can be used as part of/to form anFc portion), to an Fc portion and/or to one or more antibody parts,fragments or domains that confer one or more effector functions to thepolypeptide of the invention and/or may confer the ability to bind toone or more Fc receptors, optionally via a suitable linker or hingeregion.

Embodiment 45

A polypeptide according to any one of embodiments 2 to 37, wherein saidpolypeptide further comprises a second immunoglobulin single variabledomain, preferably a second anti-CX3CR1 immunoglobulin single variabledomain.

Embodiment 46

A polypeptide according to embodiment 45, wherein said first and saidsecond immunoglobulin single variable domains are covalently linked by alinker peptide.

Embodiment 47

A polypeptide according to embodiment 45 or 46, wherein said secondimmunoglobulin single variable domains essentially consist of fourframework regions (FR1 to FR4) and three complementary determiningregions (CDR1 to CDR3).

Embodiment 48

A polypeptide according to any one of embodiments 45 to 47, wherein saidfirst and said second immunoglobulin single variable domains areantibody domains.

Embodiment 49

A polypeptide according to any one of embodiments 45 to 48, wherein saidfirst and second immunoglobulin single variable domains are a VH, VL,VHH, camelized VH, or VHH that is optimized for stability, potency,manufacturability and/or similarity to human framework regions.

Embodiment 50

A polypeptide according to any one of embodiments 45 to 49, wherein saidCDR1 to CDR3 of said second immunoglobulin single variable domain areset forth in any one of embodiments 11 to 21.

Embodiment 51

A polypeptide according to any one of embodiments 45 to 50, wherein saidfirst and said second immunoglobulin single variable domains comprisethe same CDR3.

Embodiment 52

A polypeptide according to embodiment 51, wherein said CDR3 is set forthin any one of embodiment 11 to 13.

Embodiment 53

A polypeptide according to any one of embodiments 45 to 53, wherein saidfirst and said second immunoglobulin single variable domains comprisethe same CDR1, CDR2 and CDR3.

Embodiment 54

A polypeptide according to embodiment 53, wherein said CDR1 to CDR3 areset forth in any one of embodiments 11 to 21.

Embodiment 55

A polypeptide according to any one of embodiments 45 to 54, wherein saidfirst and said second immunoglobulin single variable domains comprisethe same VHH domain.

Embodiment 56

A The polypeptide according to any one of embodiments 45 to 55, whereinsaid VHH domain is set forth in any one of embodiments 22 to 37.

Embodiment 57

A polypeptide comprising a first immunoglobulin single variable domaincomprising the CDR1, CDR2 and CDR3 set forth SEQ ID NO's: 141, 164 and186 or SEQ ID NO's: 141, 162 and 186 and a second immunoglobulin singlevariable domain as set forth in any one of embodiments 2 to 37.

Such a polypeptide may in particular be a polypeptide according to anyof embodiments 45 to 56.

Embodiment 58

A polypeptide according to embodiment 57, wherein said firstimmunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3set forth in SEQ ID NO's: 213, 214 and 186, SEQ ID NO's: 213, 221 and186 or SEQ ID NO's: 141, 162 and 186.

Embodiment 59

A polypeptide according to embodiment 57 or 58, wherein said secondimmunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3set forth SEQ ID NO's: 141, 164 and 186 or SEQ ID NO's: 141, 162 and186.

Embodiment 60

A polypeptide according to embodiment 57 or 58, wherein said secondimmunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3set forth in: SEQ ID NO's: 213, 214 and 186, SEQ ID NO's: 213, 221 and186 or SEQ ID NO's: 141, 162 and 186.

Embodiment 61

A polypeptide comprising a first immunoglobulin single variable domain,wherein said first immunoglobulin single variable domain is a VHH domaincomprising the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and asecond immunoglobulin single variable domain according to any one ofembodiments 2 to 37.

Such a polypeptide may in particular be a polypeptide according to anyof embodiments 45 to 60.

Embodiment 62

A polypeptide according to embodiment 61, wherein said firstimmunoglobulin single variable domain is a VHH domain comprising thesequence set forth in any one of SEQ ID NO: 121-140 or 222-224.

Embodiment 63

A polypeptide according to embodiment 61 or 62, wherein said secondimmunoglobulin single variable domain is a VHH domain comprising thesequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment 64

A polypeptide according to embodiment 63, wherein said secondimmunoglobulin single variable domain is a VHH domain comprising thesequence set forth in any one of SEQ ID NO: 121-140 or 222-224.

Embodiment 65

A polypeptide according to any one of embodiments 45 to 64, wherein thepolypeptide further comprises a half-life extending moiety.

Embodiment 66

A polypeptide according to embodiment 65, wherein said half-lifeextending moiety is covalently linked to said polypeptide and isselected from the group consisting of an albumin binding moiety, such asan anti-albumin immunoglobulin domain, a transferrin binding moiety,such as an anti-transferrin immunoglobulin domain, a polyethylene glycolmolecule, a recombinant polyethylene glycol molecule, human serumalbumin, a fragment of human serum albumin, an albumin binding peptideor a Fc domain.

Embodiment 67

A polypeptide according to embodiment 66, wherein said half-lifeextending moiety consists of an anti-albumin immunoglobulin singlevariable domain.

Embodiment 68

A polypeptide according to embodiment 67, wherein the immunoglobulinsingle variable domain is selected from a VHH domain, a humanized VHHdomain, a camelized VH domain, a domain antibody, a single domainantibody and/or “dAb”s.

Embodiment 69

A polypeptide according to embodiment 68, wherein the anti-albuminimmunoglobulin single variable domain is selected from SEQ ID NO's:230-232.

Embodiment 70

A polypeptide according to any one of embodiments 45 to 64, wherein saidpolypeptide is linked to an Fc portion (such as a human Fc, for exampleas set forth in SEQ ID NO: 252), optionally via a suitable linker orhinge region.

Embodiment 71

A polypeptide according to any one of embodiments 45 to 66, wherein saidpolypeptide is further linked to one or more constant domains (forexample, 2 or 3 constant domains that can be used as part of/to form anFc portion), to an Fc portion and/or to one or more antibody parts,fragments or domains that confer one or more effector functions to thepolypeptide of the invention and/or may confer the ability to bind toone or more Fc receptors, optionally via a suitable linker or hingeregion.

Embodiment 72

A polypeptide comprising the amino acid sequence of any one of SEQ IDNO: 225-227.

Embodiment 73

A polypeptide comprising the amino acid sequence of any one of SEQ IDNO: 249 or 277-281.

Embodiment 74

A polypeptide comprising the amino acid sequence of any one of SEQ IDNO: 257-262.

Embodiment 75

A polypeptide comprising the amino acid sequence of any one of SEQ IDNO: 253 or 254.

Embodiment 76

A polypeptide comprising the amino acid sequence of any one of SEQ IDNO: 263 or 266.

Embodiment 77

A polypeptide comprising the amino acid sequence of any one of SEQ IDNO: 267-276 and 282.

Embodiment 78

A nucleic acid molecule comprising a region encoding a polypeptideaccording to any one of embodiments 1 to 77.

Embodiment 79

An expression vector comprising a nucleic acid molecule according toembodiment 78.

Embodiment 80

A host cell carrying an expression vector comprising a nucleic acidmolecule, said nucleic acid molecule comprising a region encoding apolypeptide according to any one of embodiments 1 to 77, wherein saidhost cell is capable of expressing a polypeptide according to any one ofembodiments 1 to 77, and wherein said host cell is a prokaryotic or aeukaryotic cell.

Embodiment 81

A pharmaceutical composition comprising (i) as the active ingredient,one or more polypeptides according to any one of embodiments 1 to 77,and (ii) a pharmaceutically acceptable carrier, and optionally (iii) adiluent, excipient, adjuvant and/or stabilizer.

Embodiment 82

A method of manufacturing a polypeptide according to any one ofembodiments 1 to 77, comprising the steps of

-   -   culturing a host cell under conditions that allow expression of        a polypeptide according to any one of embodiments 1 to 77,    -   wherein said host cell carrying an expression vector comprising        a nucleic acid molecule, said nucleic acid molecule comprising a        region encoding a polypeptide according to any one of        embodiments 1 to 77, and wherein said host cell is a prokaryotic        or a eukaryotic cell;    -   recovering said polypeptide; and    -   purifying said polypeptide.

Embodiment 83

A method of using a polypeptide according to any one of embodiments 1 to77 for the treatment, prevention or alleviation of a disease, disorderor condition, in particular in a human being.

Embodiment 84

The method of embodiment 83, wherein said disease, disorder or conditionis a CX3CR1-associated disease, disorder or condition.

Embodiment 85

The method of embodiment 83, wherein said disease, disorder or conditionis atherosclerosis.

Embodiment 86

An injectable pharmaceutical composition comprising the polypeptideaccording to any one of embodiments 1 to 77, said composition beingsuitable for intravenous or subcutaneous injection in a human being.

Embodiment 87

A method for preventing and/or treating a disease or disorder that isassociated with CX3CR1, wherein said method comprises administering to asubject in need thereof a pharmaceutically active amount of at least onepolypeptide according to any one of embodiments 1 to 77.

Embodiment 88

A method of embodiment 85, further comprising administering anadditional therapeutic agent selected from the group consisting of astatin, an antiplatelet, an anticoagulant, an antidiabetic and anantihypertensive.

Embodiment 89

A method for inhibiting the binding of CX3CR1 to fractalkine in amammalian cell, comprising administering to the cell a polypeptideaccording to any one of embodiments 1 to 77, whereby signaling mediatedby the fractalkine is inhibited.

Embodiment 90

A method for detecting and/or quantifying CX3CR1 levels in a biologicalsample by contacting the sample with a polypeptide according to any oneof embodiments 1 to 77 and detecting binding of the polypeptide withCX3CR1.

Embodiment 91

A method for diagnosing an CX3CR1-associated disorder or for determiningif a subject has an increased risk of developing an CX3CR1-associateddisorder, wherein the method comprises contacting a biological samplefrom a subject with a polypeptide according to any one of embodiments 1to 77 and detecting binding of the polypeptide to CX3CR1 to determinethe expression or concentration of CX3CR1.

Embodiment 92

A polypeptide according to any one of embodiments 1 to 77 for use in thetreatment, prevention or alleviation of a disease, disorder orcondition, in a human being.

Embodiment 93

The polypeptide for use according to embodiment 92, wherein the disease,disorder or condition is a CX3CR1-associated disease, disorder orcondition.

Embodiment 94

The polypeptide for use according to embodiment 92, wherein the disease,disorder or condition is selected from cardio- and cerebrovascularatherosclerotic disorders, peripheral artery disease, restenosis,diabetic nephropathy, glomerulomephritis, human crescenticglomerulonephritis, IgA nephropathy, membranous nephropathy, lupusnephritis, vasculitis including Henoch-Schonlein purpura and Wegener'sgranulomatosis, rheumatoid arthritis, osteoarthritis, allograftrejection, systemic sclerosis, neurodegenerative disorders anddemyelinating disease, multiple sclerosis (MS), Alzheimer's disease,pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatorypain, or cancer.

Embodiment 95

The polypeptide for use according to embodiment 92, wherein the disease,disorder or condition is atherosclerosis.

Embodiment 96

Use of a polypeptide according to any of embodiments 1 to 77 for themanufacture of a medicament for the treatment, prevention or alleviationof a disease, disorder or condition, in a human being.

Embodiment 97

The method according to embodiment 87, wherein the disease or disorderis selected from cardio- and cerebrovascular atherosclerotic disorders,peripheral artery disease, restenosis, diabetic nephropathy,glomerulonephritis, human crescentic glomerulonephritis, IgAnephropathy, membranous nephropathy, lupus nephritis, vasculitisincluding Henoch-Schonlein purpura and Wegener's granulomatosis,rheumatoid arthritis, osteoarthritis, allograft rejection, systemicsclerosis, neurodegenerative disorders and demyelinating disease,multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such asCOPD, asthma, neuropathic pain, inflammatory pain, or cancer.

Embodiment 98

The method according to embodiment 87, wherein the disease, disorder orcondition is atherosclerosis.

Embodiment 99

A diagnostic kit or diagnostic method comprising a polypeptide accordingto any one of embodiments 1 to 77, or the use thereof.

Embodiment 100

A diagnostic kit or diagnostic method according to embodiment 99, forthe diagnosis of at least one of cardio- and cerebrovascularatherosclerotic disorders, peripheral artery disease, restenosis,diabetic nephropathy, glomerulomephritis, human crescenticglomerulonephritis, IgA nephropathy, membranous nephropathy, lupusnephritis, vasculitis including Henoch-Schonlein purpura and Wegener'sgranulomatosis, rheumatoid arthritis, osteoarthritis, allograftrejection, systemic sclerosis, neurodegenerative disorders anddemyelinating disease, multiple sclerosis (MS), Alzheimer's disease,pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatorypain, or cancer.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The above and other aspects and embodiments of the invention will becomeclear from the further description herein, in which:

a) Unless indicated or defined otherwise, all terms used have theirusual meaning in the art, which will be clear to the skilled person.Reference is for example made to the standard handbooks, such asSambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd Ed.),Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, “GenesIV”, Oxford University Press, New York, (1990), and Roitt et al.,“Immunology” (2^(nd) Ed.), Gower Medical Publishing, London, N.Y.(1989), as well as to the general background art cited herein;Furthermore, unless indicated otherwise, all methods, steps, techniquesand manipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks, to the general background art referred to above andto the further references cited therein;

b) Unless indicated otherwise, the terms “immunoglobulin” and“immunoglobulin sequence”—whether used herein to refer to a heavy chainantibody or to a conventional 4-chain antibody—are used as general termsto include both the full-size antibody, the individual chains thereof,as well as all parts, domains or fragments thereof (including but notlimited to antigen-binding domains or fragments such as VHH domains orVH/VL domains, respectively). In addition, the term “sequence” as usedherein (for example in terms like “immunoglobulin sequence”, “antibodysequence”, “(single) variable domain sequence”, “VHH sequence” or“protein sequence”), should generally be understood to include both therelevant amino acid sequence as well as nucleic acid sequences ornucleotide sequences encoding the same, unless the context requires amore limited interpretation;

c) The term “domain” (of a polypeptide or protein) as used herein refersto a folded protein structure which has the ability to retain itstertiary structure independently of the rest of the protein. Generally,domains are responsible for discrete functional properties of proteins,and in many cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain.

d) The term “immunoglobulin domain” as used herein refers to a globularregion of an antibody chain (such as e.g. a chain of a conventional4-chain antibody or of a heavy chain antibody), or to a polypeptide thatessentially consists of such a globular region. Immunoglobulin domainsare characterized in that they retain the immunoglobulin foldcharacteristic of antibody molecules, which consists of a 2-layersandwich of about 7 antiparallel beta-strands arranged in twobeta-sheets, optionally stabilized by a conserved disulphide bond.

e) The term “immunoglobulin variable domain” as used herein means animmunoglobulin domain essentially consisting of four “framework regions”which are referred to in the art and hereinbelow as “framework region 1”or “FR1”; as “framework region 2” or“FR2”; as “framework region 3” or“FR3”; and as “framework region 4” or “FR4”, respectively; whichframework regions are interrupted by three “complementarity determiningregions” or “CDRs”, which are referred to in the art and hereinbelow as“complementarity determining region 1” or “CDR1”; as “complementaritydetermining region 2” or “CDR2”; and as “complementarity determiningregion 3” or “CDR3”, respectively. Thus, the general structure orsequence of an immunoglobulin variable domain can be indicated asfollows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulinvariable domain(s) that confer specificity to an antibody for theantigen by carrying the antigen-binding site.

f) The terms “immunoglobulin single variable domain” and “singlevariable domain” as used herein mean an immunoglobulin variable domainwhich is capable of specifically binding to an epitope of the antigenwithout pairing with an additional variable immunoglobulin domain. Oneexample of immunoglobulin single variable domains in the meaning of thepresent invention are “domain antibodies”, such as the immunoglobulinsingle variable domains VH and VL (VH domains and VL domains). Anotherexample of immunoglobulin single variable domains are “VHH domains” (orsimply “VHHs”) from camelids, as defined hereinafter.

In view of the above definition, the antigen-binding domain of aconventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgEmolecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, anFv fragment such as a disulphide linked Fv or a scFv fragment, or adiabody (all known in the art) derived from such conventional 4-chainantibody, would normally not be regarded as an immunoglobulin singlevariable domain, as, in these cases, binding to the respective epitopeof an antigen would normally not occur by one (single) immunoglobulindomain but by a pair of (associating) immunoglobulin domains such aslight and heavy chain variable domains, i.e. by a VH-VL pair ofimmunoglobulin domains, which jointly bind to an epitope of therespective antigen.

f1) “VHH domains”, also known as VHHs, V_(H)H domains, VHH antibodyfragments, and VHH antibodies, have originally been described as theantigen binding immunoglobulin (variable) domain of “heavy chainantibodies” (i.e. of “antibodies devoid of light chains”;Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C,Songa E B, Bendahman N, Hamers R.: “Naturally occurring antibodiesdevoid of light chains”; Nature 363, 446-448 (1993)). The term “VHHdomain” has been chosen in order to distinguish these variable domainsfrom the heavy chain variable domains that are present in conventional4-chain antibodies (which are referred to herein as “V_(H) domains” or“VH domains”) and from the light chain variable domains that are presentin conventional 4-chain antibodies (which are referred to herein as“V_(L) domains” or “VL domains”). VHH domains can specifically bind toan epitope without an additional antigen binding domain (as opposed toVH or VL domains in a conventional 4-chain antibody, in which case theepitope is recognized by a VL domain together with a VH domain). VHHdomains are small, robust and efficient antigen recognition units formedby a single immunoglobulin domain.

In the context of the present invention, the terms VHH domain, VHH,V_(H)H domain, VHH antibody fragment, VHH antibody, as well as“Nanobody®” and “Nanobody® domain” (“Nanobody” being a trademark of thecompany Ablynx N.V.; Ghent; Belgium) are used interchangeably and arerepresentatives of immunoglobulin single variable domains (having thestructure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to anepitope without requiring the presence of a second immunoglobulinvariable domain), and which are distinguished from VH domains by theso-called “hallmark residues”, as defined in e.g. WO2009/109635, FIG. 1.

The amino acid residues of a VHH domain are numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to VHH domains fromCamelids, as shown e.g. in FIG. 2 of Riechmann and Muyldermans, J.Immunol. Methods 231, 25-38 (1999). According to this numbering,

-   -   FR1 comprises the amino acid residues at positions 1-30,    -   CDR1 comprises the amino acid residues at positions 31-35,    -   FR2 comprises the amino acids at positions 36-49,    -   CDR2 comprises the amino acid residues at positions 50-65,    -   FR3 comprises the amino acid residues at positions 66-94,    -   CDR3 comprises the amino acid residues at positions 95-102, and    -   FR4 comprises the amino acid residues at positions 103-113.

However, it should be noted that—as is well known in the art for V_(H)domains and for VHH domains—the total number of amino acid residues ineach of the CDRs may vary and may not correspond to the total number ofamino acid residues indicated by the Kabat numbering (that is, one ormore positions according to the Kabat numbering may not be occupied inthe actual sequence, or the actual sequence may contain more amino acidresidues than the number allowed for by the Kabat numbering). This meansthat, generally, the numbering according to Kabat may or may notcorrespond to the actual numbering of the amino acid residues in theactual sequence.

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner to VHHdomains, are known in the art. However, in the present description,claims and FIGURES, the numbering according to Kabat and applied to VHHdomains as described above will be followed, unless indicated otherwise.

The total number of amino acid residues in a VHH domain will usually bein the range of from 110 to 120, often between 112 and 115. It shouldhowever be noted that smaller and longer sequences may also be suitablefor the purposes described herein.

Further structural characteristics and functional properties of VHHdomains and polypeptides containing the same can be summarized asfollows:

VHH domains (which have been “designed” by nature to functionally bindto an antigen without the presence of, and without any interaction with,a light chain variable domain) can function as a single, relativelysmall, functional antigen-binding structural unit, domain orpolypeptide. This distinguishes the VHH domains from the VH and VLdomains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or immunoglobulin single variable domains, but need to becombined in some form or another to provide a functional antigen-bindingunit (as in for example conventional antibody fragments such as Fabfragments; in scFv's, which consist of a VH domain covalently linked toa VL domain).

Because of these unique properties, the use of VHH domains—either aloneor as part of a larger polypeptide—offers a number of significantadvantages over the use of conventional VH and VL domains, scFv's orconventional antibody fragments (such as Fab- or F(ab′)2-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spacial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   VHH domains can be expressed from a single gene and require no        post-translational folding or modifications;    -   VHH domains can easily be engineered into multivalent and        multispecific formats (as further discussed herein);    -   VHH domains are highly soluble and do not have a tendency to        aggregate (as with the mouse-derived antigen-binding domains        described by Ward et al., Nature 341: 544-546 (1989));    -   VHH domains are highly stable to heat, pH, proteases and other        denaturing agents or conditions and, thus, may be prepared,        stored or transported without the use of refrigeration        equipments, conveying a cost, time and environmental savings;    -   VHH domains are easy and relatively cheap to prepare, even on a        scale required for production. For example, VHH domains and        polypeptides containing the same can be produced using microbial        fermentation (e.g. as further described below) and do not        require the use of mammalian expression systems, as with for        example conventional antibody fragments;    -   VHH domains are relatively small (approximately 15 kDa, or 10        times smaller than a conventional IgG) compared to conventional        4-chain antibodies and antigen-binding fragments thereof, and        therefore    -   show high(er) penetration into tissues and    -   can be administered in higher doses than such conventional        4-chain antibodies and antigen-binding fragments thereof;    -   VHH domains can show so-called cavity-binding properties (inter        alia due to their extended CDR3 loop, compared to conventional        VH domains) and can therefore also access targets and epitopes        not accessible to conventional 4-chain antibodies and        antigen-binding fragments thereof.

Methods of obtaining VHH domains binding to a specific antigen orepitope have been described earlier, e.g. in WO2006/040153 andWO2006/122786. As also described therein in detail, VHH domains derivedfrom camelids can be “humanized” by replacing one or more amino acidresidues in the amino acid sequence of the original VHH sequence by oneor more of the amino acid residues that occur at the correspondingposition(s) in a VH domain from a conventional 4-chain antibody from ahuman being. A humanized VHH domain can contain one or more fully humanframework region sequences, and, in an even more specific embodiment,can contain human framework region sequences derived from DP-29, DP-47,DP-51, or parts thereof, optionally combined with JH sequences, such asJHS.

f2) “Domain antibodies”, also known as “Dab”s, “Domain Antibodies”, and“dAbs” (the terms “Domain Antibodies” and “dAbs” being used astrademarks by the GlaxoSmithKline group of companies) have beendescribed in e.g. Ward, E. S., et al.: “Binding activities of arepertoire of single immunoglobulin variable domains secreted fromEscherichia coli”; Nature 341: 544-546 (1989); Holt, L. J. et al.:“Domain antibodies: proteins for therapy”; TRENDS in Biotechnology21(11): 484-490 (2003); and WO2003/002609.

Domain antibodies essentially correspond to the VH or VL domains ofnon-camelid mammalians, in particular human 4-chain antibodies. In orderto bind an epitope as a single antigen binding domain, i.e. withoutbeing paired with a VL or VH domain, respectively, specific selectionfor such antigen binding properties is required, e.g. by using librariesof human single VH or VL domain sequences. Domain antibodies have, likeVHHs, a molecular weight of approximately 13 to approximately 16 kDaand, if derived from fully human sequences, do not require humanizationfor e.g. therapeutical use in humans. As in the case of VHH domains,they are well expressed also in prokaryotic expression systems,providing a significant reduction in overall manufacturing cost.

Domain antibodies, as well as VHH domains, can be subjected to affinitymaturation by introducing one or more alterations in the amino acidsequence of one or more CDRs, which alterations result in an improvedaffinity of the resulting immunoglobulin single variable domain for itsrespective antigen, as compared to the respective parent molecule.Affinity-matured immunoglobulin single variable domain molecules of theinvention may be prepared by methods known in the art, for example, asdescribed by Marks et al., 1992, Biotechnology 10:779-783, or Barbas, etal., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shier et al., 1995,Gene 169:147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jacksonet al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J.Mol. Biol. 226(3): 889 896; K S Johnson and R E Hawkins, “Affinitymaturation of antibodies using phage display”, Oxford University Press1996.

f3) Furthermore, it will also be clear to the skilled person that it ispossible to “graft” one or more of the CDR's mentioned above onto other“scaffolds”, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting are known in the art.

g) The terms “epitope” and “antigenic determinant”, which can be usedinterchangeably, refer to the part of a macromolecule, such as apolypeptide, that is recognized by antigen-binding molecules, such asconventional antibodies or the polypeptides of the invention, and moreparticularly by the antigen-binding site of said molecules. Epitopesdefine the minimum binding site for an immunoglobulin, and thusrepresent the target of specificity of an immunoglobulin.

The part of an antigen-binding molecule (such as a conventional antibodyor a polypeptide of the invention) that recognizes the epitope is calleda paratope.

h) The term “biparatopic” (antigen-)binding molecule or “biparatopic”polypeptide as used herein shall mean a polypeptide comprising a firstimmunoglobulin single variable domain and a second immunoglobulin singlevariable domain as herein defined, wherein these two variable domainsare capable of binding to two different epitopes of one antigen, whichepitopes are not normally bound at the same time by one monospecificimmunoglobulin, such as e.g. a conventional antibody or oneimmunoglobulin single variable domain. Biparatopic polypeptides can becomposed of variable domains which have different epitope specificities,and do not contain mutually complementary variable domain pairs whichbind to the same epitope. The two variable domains do therefore notcompete with each other for binding to the target.

i) A polypeptide (such as an immunoglobulin, an antibody, animmunoglobulin single variable domain, a polypeptide of the invention,or generally an antigen binding molecule or a fragment thereof) that can“bind to” or “specifically bind to”, that “has affinity foe” and/or that“has specificity foe” a certain epitope, antigen or protein (or for atleast one part, fragment or epitope thereof) is said to be “against” or“directed against” said epitope, antigen or protein or is a “binding”molecule with respect to such epitope, antigen or protein, or is said tobe “anti”-epitope, “anti”-antigen or “anti”-protein (e.g anti-CX3CR1).

k) Generally, the term “specificity” refers to the number of differenttypes of antigens or epitopes to which a particular antigen-bindingmolecule or antigen-binding protein (such as an immunoglobulin, anantibody, an immunoglobulin single variable domain, or a polypeptide ofthe invention) can bind. The specificity of an antigen-binding proteincan be determined based on its affinity and/or avidity. The affinity,represented by the equilibrium constant for the dissociation of anantigen with an antigen-binding protein (KD), is a measure for thebinding strength between an epitope and an antigen-binding site on theantigen-binding protein: the lesser the value of the KD, the strongerthe binding strength between an epitope and the antigen-binding molecule(alternatively, the affinity can also be expressed as the affinityconstant (KA), which is 1/KD). As will be clear to the skilled person(for example on the basis of the further disclosure herein), affinitycan be determined in a manner known per se, depending on the specificantigen of interest. Avidity is the measure of the strength of bindingbetween an antigen-binding molecule (such as an immunoglobulin, anantibody, an immunoglobulin single variable domain, or a polypeptide ofthe invention) and the pertinent antigen. Avidity is related to both theaffinity between an epitope and its antigen binding site on theantigen-binding molecule and the number of pertinent binding sitespresent on the antigen-binding molecule.

l) Amino acid residues will be indicated according to the standardthree-letter or one-letter amino acid code, as generally known andagreed upon in the art. When comparing two amino acid sequences, theterm “amino acid difference” refers to insertions, deletions orsubstitutions of the indicated number of amino acid residues at aposition of the reference sequence, compared to a second sequence. Incase of substitution(s), such substitution(s) will preferably beconservative amino acid substitution(s), which means that an amino acidresidue is replaced with another amino acid residue of similar chemicalstructure and which has little or essentially no influence on thefunction, activity or other biological properties of the polypeptide.Such conservative amino acid substitutions are well known in the art,for example from WO 98/49185, wherein conservative amino acidsubstitutions preferably are substitutions in which one amino acidwithin the following groups (i)-(v) is substituted by another amino acidresidue within the same group: (i) small aliphatic, nonpolar or slightlypolar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar, negativelycharged residues and their (uncharged) amides: Asp, Asn, Glu and Gln;(iii) polar, positively charged residues: His, Arg and Lys; (iv) largealiphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (v)aromatic residues: Phe, Tyr and Trp. Particularly preferred conservativeamino acid substitutions are as follows:

Ala into Gly or into Ser;

Arg into Lys;

Asn into Gln or into His;

Asp into Glu;

Cys into Ser;

Gln into Asn;

Glu into Asp;

Gly into Ala or into Pro;

His into Asn or into Gln;

Ile into Leu or into Val;

Leu into Ile or into Val;

Lys into Arg, into Gln or into Glu;

Met into Leu, into Tyr or into Ile;

Phe into Met, into Leu or into Tyr;

Ser into Thr;

Thr into Ser;

Trp into Tyr;

Tyr into Trp or into Phe;

Val into Ile or into Leu.

m) A nucleic acid or polypeptide molecule is considered to be “(in)essentially isolated (form)”—for example, when compared to its nativebiological source and/or the reaction medium or cultivation medium fromwhich it has been obtained—when it has been separated from at least oneother component with which it is usually associated in said source ormedium, such as another nucleic acid, another protein/polypeptide,another biological component or macromolecule or at least onecontaminant, impurity or minor component. In particular, a nucleic acidor polypeptide molecule is considered “essentially isolated” when it hasbeen purified at least 2-fold, in particular at least 10-fold, more inparticular at least 100-fold, and up to 1000-fold or more. A nucleicacid or polypeptide molecule that is “in essentially isolated form” ispreferably essentially homogeneous, as determined using a suitabletechnique, such as a suitable chromatographical technique, such aspolyacrylamide-gelelectrophoresis;

n) “Sequence identity” between e.g. two immunoglobulin single variabledomain sequences indicates the percentage of amino acids that areidentical between these two sequences. It may be calculated ordetermined as described in paragraph f) on pages 49 and 50 ofWO08/020079. “Sequence similarity” indicates the percentage of aminoacids that either are identical or that represent conservative aminoacid substitutions.

Target Specificity

The polypeptides of the invention have specificity for human CX3CR1.Thus, the polypeptides of the invention preferably bind to human CX3CR1(SEQ ID NO: 255). In one aspect, the polypeptides of the presentinvention also bind to cynomolgus CX3CR1 (SEQ ID NO: 256).

Polypeptides of the Invention

The invention provides novel pharmaceutically active agents for theprevention, treatment, alleviation and/or diagnosis of CX3CR1 associateddiseases, disorders or conditions, such as cardiovascular diseases. Inparticular, the invention provides polypeptides which bind to humanCX3CR1 and are capable of blocking the binding of human fractalkine tohuman CX3CR1. In one aspect, the polypeptide is an immunoglobulincomprising an antigen-binding domain comprising three complementaritydetermining regions CDR1, CDR2 and CDR3, wherein said immunoglobulinbinds to human CX3CR1 and is capable of blocking the binding of humanfractalkine to human CX3CR1. In a further aspect, the polypeptidecomprises one or more anti-CX3CR1 immunoglobulin single variable domain,wherein said polypeptide is capable of blocking the binding of humanfractalkine to human CX3CR1.

In one aspect, a polypeptide of the present invention is characterizedby one or more of the following properties:

-   -   Bind with high affinity to human CX3CR1, for example at an EC₅₀        of less than or equal to 10 nM, less than or equal to 5 nM, less        than or equal to 2.5 nM or less than or equal to 1 nM, as        determined by cell binding FACS;    -   Block the binding of human fractalkine to human CX3CR1, for        example at an IC50 of less than or equal to 300 nM, or less than        or equal to 100 nM, or less than or equal to 20 nM, or less than        or equal to 10 nM, or less than or equal to 5 nM, or less than        or equal to 2.5 nM or less than or equal to 1 nM;    -   Inhibit fractalkine induced chemotaxis mediated by human CX3CR1,        for example at an IC₅₀ of less than or equal to 500 nM, or less        than or equal to 100 nM, or of less than or equal to 75 nM, or        less than or equal to 50 nM, or less than or equal to 10 nM or        less than or equal to 5 nM; the obtained efficacy of inhibition        is more than or equal to 15%, or more than or equal to 50%, or        more than or equal to 80%, or more than or equal to 95%;    -   Inhibit fractalkine induced internalization mediated by human        CX3CR1, for example at an IC₅₀ of less than or equal to 10 nM or        less than or equal to 5 nM;    -   Cross-react with cynomolgus CX3CR1, for example within 10-fold        of E/IC₅₀ for human CX3CR1 for binding and functional        inhibition.

In a further aspect, a polypeptide of the present invention furthercomprises a half-life extending moiety, for example an albumin bindingmoiety, a polyethylene glycol molecule or a Fc domain. In a furtheraspect, a polypeptide of the present invention comprises two or moreanti-CX3CR1 immunoglobulin single variable domains. In one aspect, thetwo anti-CX3CR1 immunoglobulin single variable domains are covalentlylinked by a linker peptide. In one aspect, the two anti-CX3CR1immunoglobulin single variable domains in a polypeptide of the presentinvention have the same amino acid sequence. In another aspect, the twoanti-CX3CR1 immunoglobulin single variable domains in a polypeptide ofthe present invention have different amino acid sequences. In oneaspect, a polypeptide of the present invention comprises two anti-CX3CR1immunoglobulin single variable domains and further comprises a half-lifeextending moiety, for example an albumin binding moiety, a polyethyleneglycol molecule or a Fc domain.

In one aspect, a polypeptide of the present invention comprises a firstanti-CX3CR1 immunoglobulin single variable domain covalently linked toan albumin binding moiety by a first linker peptide, wherein saidalbumin binding moiety is further covalently linked to a secondanti-CX3CR1 immunoglobulin single variable domain by a second linkerpeptide.

In one aspect, a polypeptide of the present invention comprises ananti-CX3CR1 immunoglobulin single variable domain covalently linked to aFc domain by a linker peptide. In one aspect, such polypeptidecomprising an anti-CX3CR1 immunoglobulin single variable domaincovalently linked to a Fc domain by a linker peptide is provided as adimer, for example through disulfide bridges.

Polypeptides according to the present invention are obtained asdescribed hereinbelow. In summary, single variable domains of thepresent invention were identified from a library expressing singlevariable domains (VHH) derived from a llama immunized with DNA encodinghuman CX3CR1. The phage library was panned on hCX3CR1 virallipoparticles and binding phage were screened for their ability tocompete for receptor binding with Alexa-fluor labeled fractalkine(AF-FKN). Representative single variable domains of the presentinvention are described herein in further details.

In one aspect, an immunoglobulin single variable domain of the presentinvention consists essentially of four framework regions (FR1, FR2, FR3and FR4) and three complementary determining regions (CDR1, CDR2 andCDR3). In particular, the immunoglobulin single variable domain has thestructure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In one aspect, theimmunoglobulin single variable domain is an antibody domain.

In one aspect, the CDR3 of a polypeptide of the present invention, inparticular a immunoglobulin single domain of the present invention hasthe amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5as set forth in SEQ ID NO: 197, wherein:

-   -   Xaa1 is Pro, Ala or Gly;    -   Xaa2 is Asp or Asn;    -   Xaa3 is Thr or Ser;    -   Xaa4 is Arg, Lys, Ala or Gly; and    -   Xaa5 is Tyr or Phe.

In one aspect, the CDR3 of a polypeptide of the present invention, inparticular a immunoglobulin single domain of the present invention, hasthe amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5as set forth in SEQ ID NO: 197, wherein:

-   -   Xaa1 is Pro, Ala or Gly;    -   Xaa2 is Asp or Asn;    -   Xaa3 is Thr;    -   Xaa4 is Arg or Lys; and    -   Xaa5 is Tyr.

In one aspect, the CDR3 of a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the amino acid sequence of Asp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyras set forth in SEQ ID NO: 186.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   CDR1:        -   a) has the amino acid sequence of GSIFSSNAMA (SEQ ID NO:            141); or        -   b) has an amino acid sequence that has at least 80% amino            acid identity with the amino acid sequence of SEQ ID NO:            141; or        -   c) has an amino acid sequence that has 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 141,            wherein            -   at position 2 the S has been changed into T, or G;            -   at position 6 the S has been changed into R;            -   at position 7 the N has been changed into T; and/or            -   at position 9 the M has been changed into K; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 141-145 and 213;        -   CDR2:        -   a) has the amino acid sequence of GINSVGITK (SEQ ID NO:            164); or        -   b) has an amino acid sequence that has at least 70% amino            acid identity with the amino acid sequence of SEQ ID NO:            164; or        -   c) has an amino acid sequence that has 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 164, wherein            -   at position 1 the G has been changed into A, L, V or S;            -   at position 3 the N has been changed into D, S, Q, G or                T;            -   at position 4 the S has been changed into T, K, G or P;            -   at position 5 the V has been changed into A;            -   at position 6 the G has been changed into D;            -   at position 7 the I has been changed into T, or V;            -   at position 8 the T has been changed into A; and/or            -   at position 9 the K has been changed into R; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 162-175 and 214-221; and        -   CDR3:        -   a) has the amino acid sequence of DPRRGWDTRY (SEQ ID NO:            186); or        -   b) has an amino acid sequence that has at least 70% amino            acid identity the amino acid sequence of SEQ ID NO: 186; or        -   c) has an amino acid sequence that has 3, 2, or 1 amino            acid(s) difference with the amino acid sequences of SEQ ID            NO: 186, wherein            -   at position 2 the P has been changed into A, or G;            -   at position 7 the D has been changed into N; and/or            -   at position 9 the R has been changed into K; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 186-190.

In a further aspect, a polypeptide of the present invention, inparticular a immunoglobuling single domain of the present invention, hasthe following CDR1, CDR2 and CDR3, wherein:

-   -   said CDR1 has the amino acid sequence of GRTFSSYAMG (SEQ ID NO:        146);    -   said CDR2 has an amino acid sequence that a) has at least 90%        amino acid identity with the amino acid sequence of GISGSASRKY        (SEQ ID NO: 176) or b) has the amino acid sequence of SEQ ID NO:        176 or 177; and    -   said CDR3 has the amino acid sequence of SNSYPKVQFDY (SEQ ID NO:        191).

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   said CDR1:    -   a) has the amino acid sequence of GTIFSNNAMG (SEQ ID NO: 147);        or    -   b) has an amino acid sequence that has 6, 5, 4, 3, 2, or 1 amino        acid(s) difference with the amino acid sequence of SEQ ID NO:        147, wherein        -   at position 1 the G has been changed into K, R, or A;        -   at position 2 the T has been changed into I, P, S or L;        -   at position 3 the I has been changed into V, or T;        -   at position 4 the F has been changed into L;        -   at position 5 the S has been changed into R, or D;        -   at position 6 the N has been changed into S, T, or D; and/or        -   at position 7 the N has been changed into T, or Y; or    -   c) has an amino acid sequence selected from any one of SEQ ID        NO's: 147-161;    -   said CDR2:    -   a) has the amino acid sequence of SISNSGSTN (SEQ ID NO: 179); or    -   b) has an amino acid sequences that has 4, 3, 2, or 1 amino        acid(s) difference with the amino acid sequence of SEQ ID NO:        179, wherein        -   at position 3 the S has been changed into T, or G;        -   at position 4 the N has been changed into S, or I;        -   at position 5 the S has been changed into T;        -   at position 6 the G has been changed into Y; and/or        -   at position 8 the T has been changed into A; or    -   c) has an amino acid sequence selected from any one of SEQ ID        NO's: 178-185; and    -   said CDR3:    -   a) has the amino acid sequence of DARRGWNTAY (SEQ ID NO: 192);        or    -   b) has an amino acid sequence that has at least 80% amino acid        identity with the amino acid sequence of SEQ ID NO: 192; or    -   c) has an amino acid sequence that has 2, or 1 amino acid(s)        difference with the amino acid sequence of SEQ ID NO: 192,        wherein        -   at position 2 the A has been changed into G;        -   at position 8 the T has been changed into S;        -   at position 9 the A has been changed into G; and/or        -   at position 10 the Y has been changed into F; or    -   d) has an amino acid sequence selected from any one of SEQ ID        NO's: 192-196.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   SEQ ID No: 141, 162 and 186, respectively; or    -   SEQ ID No: 141, 163 and 187, respectively; or    -   SEQ ID No: 141, 164 and 186, respectively; or    -   SEQ ID No: 141, 166 and 186, respectively; or    -   SEQ ID No: 141, 167 and 186, respectively; or    -   SEQ ID No: 141, 167 and 189, respectively; or    -   SEQ ID No: 141, 168 and 186, respectively; or    -   SEQ ID No: 141, 168 and 187, respectively; or    -   SEQ ID No: 141, 169 and 190, respectively; or    -   SEQ ID No: 141, 170 and 186, respectively; or    -   SEQ ID No: 141, 171 and 186, respectively; or    -   SEQ ID No: 141, 174 and 186, respectively; or    -   SEQ ID No: 141, 175 and 187, respectively; or    -   SEQ ID No: 142, 165 and 188, respectively; or    -   SEQ ID No: 142, 173 and 188, respectively; or    -   SEQ ID No: 143, 164 and 186, respectively; or    -   SEQ ID No: 144, 172 and 187, respectively; or    -   SEQ ID No: 145, 172 and 187, respectively; or    -   SEQ ID No: 141, 214 and 186, respectively; or    -   SEQ ID No: 141, 215 and 186, respectively; or    -   SEQ ID No: 141, 216 and 186, respectively; or    -   SEQ ID No: 141, 217 and 186, respectively; or    -   SEQ ID No: 141, 218 and 186, respectively; or    -   SEQ ID No: 141, 219 and 186, respectively; or    -   SEQ ID No: 141, 220 and 186, respectively; or    -   SEQ ID No: 213, 221 and 186, respectively; or    -   SEQ ID No: 213, 214 and 186, respectively.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   SEQ ID No: 146, 176 and 191, respectively; or    -   SEQ ID No: 146, 177 and 191, respectively.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   SEQ ID No: 147, 178 and 192, respectively; or    -   SEQ ID No: 147, 179 and 192, respectively; or    -   SEQ ID No: 147, 179 and 194, respectively; or    -   SEQ ID No: 148, 179 and 193, respectively; or    -   SEQ ID No: 149, 179 and 192, respectively; or    -   SEQ ID No: 149, 180 and 192, respectively; or    -   SEQ ID No: 149, 181 and 192, respectively; or    -   SEQ ID No: 149, 183 and 192, respectively; or    -   SEQ ID No: 149, 185 and 192, respectively; or    -   SEQ ID No: 150, 179 and 194, respectively; or    -   SEQ ID No: 150, 182 and 194, respectively; or    -   SEQ ID No: 151, 179 and 193, respectively; or    -   SEQ ID No: 151, 182 and 194, respectively; or    -   SEQ ID No: 151, 184 and 196, respectively; or    -   SEQ ID No: 152, 179 and 195, respectively; or    -   SEQ ID No: 153, 179 and 194, respectively; or    -   SEQ ID No: 154, 182 and 194, respectively; or    -   SEQ ID No: 155, 179 and 195, respectively; or    -   SEQ ID No: 156, 181 and 192, respectively; or    -   SEQ ID No: 157, 179 and 194, respectively; or    -   SEQ ID No: 158, 179 and 192, respectively; or    -   SEQ ID No: 159, 178 and 192, respectively; or    -   SEQ ID No: 160, 179 and 194, respectively; or    -   SEQ ID No: 161, 179 and 194, respectively.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the CDR1, CDR2 and CDR3 set forth in:

-   -   SEQ ID NO's: 141, 164 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the CDR1, CDR2 and CDR3 set forth in:

-   -   SEQ ID NO's: 213, 214 and 186; or    -   SEQ ID NO's: 213, 221 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

Representative polypeptides of the present invention having the CDRsdescribed above are shown in Tables 1, 2, 3 (representative polypeptidesof families 101, 9 and 13, respectively) and 4 (representativepolypeptides of optimized variants of family 101.

TABLE 1  Family 101 SEQ SEQ SEQ Nanobody SEQ CDR1* CDR1 CDR2* CDR2 CDR3*CDR3 CX3CR1BIIPMP66B02 1 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54A12 2 GSIFSSNA 141 VINSVGIT 163 DARRGW 187 MA K DTRYCX3CR1BIIPMP54A3 3 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54A4 4 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54A5 5 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54A7 6 GTIFSSNA 142 GINSVDIT 165 DPRRGW 188 MA K NTRYCX3CR1BIIPMP54B1 7 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54B2 8 GTIFSSNA 142 GINSVDIT 165 DPRRGW 188 MA K NTRYCX3CR1BIIPMP54B3 9 GSIFSSNA 141 AINSVGIT 166 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54B5 10 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54D5 11 GSIFSSNA 141 LINSVGIT 167 DGRRGW 189 MA K DTRYCX3CR1BIIPMP54D8 12 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54F6 13 GSIFSSNA 141 AINSVGIT 166 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54G3 14 GSIFSSNA 141 LINSVGIT 167 DPRRGW 186 MA K DTRYCX3CR1BIIPMP54H1 15 GTIFSSNA 142 GINSVDIT 165 DPRRGW 188 MA K NTRYCX3CR1BIIPMP54H4 16 GSIFSSNA 141 VINSVGIT 163 DARRGW 187 MA K DTRYCX3CR1BIIPMP61F10 17 GTIFSSNA 142 GINSVDIT 165 DPRRGW 188 MA K NTRYCX3CR1BIIPMP61D1 18 GSIFSSNA 141 LINSVGIT 167 DPRRGW 186 MA K DTRYCX3CR1BIIPMP61D5 19 GSIFSSNA 141 LINSVGIT 167 DPRRGW 186 MA K DTRYCX3CR1BIIPMP61E2 20 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP61F11 21 GSIFSSNA 141 AINSVGIT 166 DPRRGW 186 MA K DTRYCX3CR1BIIPMP61G2 22 GSIFSSNA 141 LINSVGIT 167 DPRRGW 186 MA K DTRYCX3CR1BIIPMP61G3 23 GSIFSSNA 141 AINSVGIT 166 DPRRGW 186 MA K DTRYCX3CR1BIIPMP61G4 24 GSIFSSNA 141 AINSVGIT 166 DPRRGW 186 MA K DTRYCX3CR1BIIPMP61F4 25 GSIFSSNA 141 VINTVGIT 168 DARRGW 187 MA K DTRYCX3CR1BIIPMP61A11 26 GSIFSSNA 141 VINSVGIT 163 DARRGW 187 MA K DTRYCX3CR1BIIPMP61B2 27 GSIFSSNA 141 VINTVGIT 168 DARRGW 187 MA K DTRYCX3CR1BIIPMP61C9 28 GSIFSSNA 141 LIDSAGIT 169 DARRGW 190 MA K NTKYCX3CR1BIIPMP65H02 29 GSIFSSNA 141 AINSVGIT 166 DPRRGW 186 MA K DTRYCX3CR1BIIPMP65E11 30 GSIFSSNA 141 GINSVGIA 170 DPRRGW 186 MA K DTRYCX3CR1BIIPMP65E10 31 GSIFSSNA 143 GINSVGIT 164 DPRRGW 186 KA K DTRYCX3CR1BIIPMP65E05 32 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP65B11 33 GSIFSSNA 141 VINKVGIT 171 DPRRGW 186 MA K DTRYCX3CR1BIIPMP65B07 34 GSIFSSNA 141 AINSVGIT 166 DPRRGW 186 MA K DTRYCX3CR1BIIPMP65B09 35 GSIFSRNA 144 SINSVGIT 172 DARRGW 187 MA K DTRYCX3CR1BIIPMP65H01 36 GGIFSRN 145 SINSVGIT 172 DARRGW 187 AMA K DTRYCX3CR1BIIPMP65G07 37 GTIFSSNA 142 GINSVDIT 173 DPRRGW 188 MA R NTRYCX3CR1BIIPMP66H08 38 GSIFSSNA 141 LINSVGIT 167 DPRRGW 186 MA K DTRYCX3CR1BIIPMP66H04 39 GSIFSSNA 141 AINSVGIT 166 DPRRGW 186 MA K DTRYCX3CR1BIIPMP66F02 40 GSIFSSNA 141 LINSVGIT 167 DPRRGW 186 MA K DTRYCX3CR1BIIPMP66E11 41 GSIFSSNA 141 AINSVGTT 174 DPRRGW 186 MA K DTRYCX3CR1BIIPMP66D10 42 GSIFSSNA 141 LINSVGIT 167 DPRRGW 186 MA K DTRYCX3CR1BIIPMP66D08 43 GSIFSSNA 141 GINSVGIT 164 DPRRGW 186 MA K DTRYCX3CR1BIIPMP66A04 44 GSIFSSNA 141 LINSVGIT 167 DPRRGW 186 MA K DTRYCX3CR1BIIPMP66D04 45 GTIFSSNA 142 GINSVDIT 165 DPRRGW 188 MA K NTRYCX3CR1BIIPMP66D02 46 GSIFSSNA 141 VINSVGIT 163 DARRGW 187 MA K DTRYCX3CR1BIIPMP66D06 47 GSIFSSNA 141 SIDSVGIT 175 DARRGW 187 MA K DTRYCX3CR1BIIPMP66G01 48 GSIFSSNA 141 LINSVGIT 167 DGRRGW 189 MA K DTRY *CDRsequences were determined according to Antibody Engineering, vol 2 byKonetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Thesequence numbers in the table (SEQ) refer to the sequences in thesequence listing of the instant application.

TABLE 2  Family 9 SEQ SEQ SEQ Nanobody SEQ CDR1* CDR1 CDR2* CDR2 CDR3*CDR3 CX3CR1BIIPMP11H11 49 GRTFS 146 GISGS 176 SNSYPKV 191 SYAMG ASRKYQFDY CX3CR1BIIPMP12B6 50 GRTFS 146 GISGS 176 SNSYPKV 191 SYAMG ASRKYQFDY CX3CR1BIIPMP12G9 51 GRTFS 146 GISGS 177 SNSYPKV 191 SYAMG GSRKYQFDY CX3CR1BIIPMP15G11 52 GRTFS 146 GISGS 177 SNSYPKV 191 SYAMG GSRKYQFDY *CDR sequences were determined according to Antibody Engineering,vol 2 by Konetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin,2010. The sequence numbers in the table (SEQ) refer to the sequences inthe sequence listing of the instant application.

TABLE 3  Family 13 SEQ SEQ SEQ Nanobody SEQ CDR1* CDR1 CDR2* CDR2 CDR3*CDR3 CX3CR1BIIPMP18E6 53 GTIFSNNA 147 SISSSGST 178 DARRGW 192 MG N NTAYCX3CR1BIIPMP12C2 54 GTIFSNTA 148 SISNSGST 179 DARRGW 193 MG N NSGYCX3CR1BIIPMP18A10 55 GIIFSNNA 149 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18A2 56 GIIFSNNA 149 SIGSTYST 180 DARRGW 192 MG N NTAYCX3CR1BIIPMP18A8 57 RTIFRSNA 150 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP18A9 58 GIIFSNNA 149 SISSTYST 181 DARRGW 192 MG N NTAYCX3CR1BIIPMP18B7 59 GTIFRSNA 151 SISNSGST 179 DARRGW 193 MG N NSGYCX3CR1BIIPMP18B9 60 GTIFSNNA 147 SISSSGST 178 DARRGW 192 MG N NTAYCX3CR1BIIPMP18C6 61 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18C9 62 GIIFSNNA 149 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18D1 63 GIIFSNNA 149 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18D10 64 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18D12 65 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18F1 66 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18F5 67 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18F6 68 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18F9 69 GTIFRTNA 152 SISNSGST 179 DGRRGW 195 MG N NTGYCX3CR1BIIPMP18G5 70 RTIFRSNA 150 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP18H1 71 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18H10 72 KTIFRSNA 153 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP18H7 73 GIIFSNNA  149 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP18H9 74 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP20B3 75 GIIFSNNA  149 SIGSTYST 180 DARRGW 192 MG N NTAYCX3CR1BIIPMP20C12 76 GTIFRSNA 151 SISNSGST 179 DARRGW 193 MG N NSGYCX3CR1BIIPMP20C3 77 GIIFSNNA  149 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP2006 78 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP20D8 79 GTTFRSN 154 SITNSGST 182 DARRGW 194 AMG N NTGYCX3CR1BIIPMP20E11 80 RTIFRSNA 150 SITNSGST 182 DARRGW 194 MG N NTGYCX3CR1BIIPMP20E5 81 GTIFSNNA 147 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP20F3 82 GTIFSNNA 147 SISSSGST 178 DARRGW 192 MG N NTAYCX3CR1BIIPMP20F4 83 ATIFRSNA  155 SISNSGST 179 DGRRGW 195 MG N NTGYCX3CR1BIIPMP20F5 84 ATIFRSNA  155 SISNSGST 179 DGRRGW 195 MG N NTGYCX3CR1BIIPMP21B6 85 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP24A12 86 GIIFSNNA  149 SISNSGSA 183 DARRGW 192 MG N NTAYCX3CR1BIIPMP24A6 87 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP24B9 88 GTIFRSNA 151 SISISGST 184 DARRGW 196 MG N NTGFCX3CR1BIIPMP24D3 89 GIIFSNNA  149 SISSTYST 181 DARRGW 192 MG N NTAYCX3CR1BIIPMP24F7 90 GLIFSNNA 156 SISSTYST 181 DARRGW 192 MG N NTAYCX3CR1BIIPMP28B4 91 ATIFRSNA 155 SISNSGST 179 DGRRGW 195 MG N NTGYCX3CR1BIIPMP28F1 92 GIIFSNNA 149 SIGSTYST 180 DARRGW 192 MG N NTAYCX3CR1BIIPMP28F6 93 GIIFSNNA 149 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP28F9 94 GTIFSNNA 147 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP29A5 95 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP29D5 96 GTIFRSNA 151 SISNSGST 179 DARRGW 193 MG N NSGYCX3CR1BIIPMP29E3 97 KTIFRSNA 153 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP29E7 98 KTIFRSNA 153 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP29G10 99 GTIFRSNA 151 SITNSGST 182 DARRGW 194 MG N NTGYCX3CR1BIIPMP29G7 100 GIIFSNNA  149 SITNTGST 185 DARRGW 192 MG N NTAYCX3CR1BIIPMP29H1 101 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP37A8 102 RTIFRSNA 150 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP37B9 103 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP37C12 104 GSIFRSNA 157 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP3707 105 RTIFSNNA 158 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP37D9 106 GTVFSNN 159 SISSSGST 178 DARRGW 192 AMG N NTAYCX3CR1BIIPMP37E12 107 KPIFRSNA 160 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP41B10 108 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP41B11 109 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAYCX3CR1BIIPMP41B8 110 GIIFSNNA 149 SIGSTYST 180 DARRGW 192 MG N NTAYCX3CR1BIIPMP41C10 111 RTIFRSNA 150 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP41F9 112 GIIFSNNA  149 SIGSTYST 180 DARRGW 192 MG N NTAYCX3CR1BIIPMP41H10 113 GLTLDDY 161 SISNSGST 179 DARRGW 194 AMG N NTGYCX3CR1BIIPMP46B5 114 RTIFRSNA 150 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP46D3 115 GTIFSNNA 147 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP46H5 116 GIIFSNNA  149 SISSTYST 181 DARRGW 192 MG N NTAYCX3CR1BIIPMP48B8 117 KTIFRSNA 153 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP48D11 118 RTIFRSNA 150 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP48G8 119 RTIFRSNA 150 SISNSGST 179 DARRGW 194 MG N NTGYCX3CR1BIIPMP48H9 120 GTIFSNNA 147 SISNSGST 179 DARRGW 192 MG N NTAY *CDRsequences were determined according to Antibody Engineering, vol 2 byKonetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Thesequence numbers in the table (SEQ) refer to the sequences in thesequence listing of the instant application.

TABLE 4  Optimized variants SEQ SEQ SEQ Nanobody SEQ CDR1 CDR1 CDR2 CDR2CDR3 CDR3 CX3CR1BIIPMP66B02   1   GSIFSSNA 141 AINSVGVT 162 DPRRGW 186MA K DTRY CX3CR1BII043 121 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA KDTRY CX3CR1BII045 122 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII047 123 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII048 124 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII049 125 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII050 126 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII061 127 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII056 128 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII057 129 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII060 130 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRYCX3CR1BII065 131 GSIFSSNA 141 AISSVGVT 214 DPRRGW 186 MA K DTRYCX3CR1BII067 132 GSIFSSNA 141 AIQSVGVT 215 DPRRGW 186 MA K DTRYCX3CR1BII068 133 GSIFSSNA 141 AIGSVGVT 216 DPRRGW 186 MA K DTRYCX3CR1BII074 134 GSIFSSNA 141 AITSVGVT 217 DPRRGW 186 MA K DTRYCX3CR1BII118 135 GSIFSSNA 141 AINTVGVT 218 DPRRGW 186 MA K DTRYCX3CR1BII129 136 GSIFSSNA 141 AINGVGV 219 DPRRGW 186 MA TK DTRYCX3CR1BII158 137 GSIFSSNA 141 AINPVGVT 220 DPRRGW 186 MA K DTRYCX3CR1BII306 138 GSIFSSTA 213 AISSVGVT 214 DPRRGW 186 MA K DTRYCX3CR1BII307 139 GSIFSSTA 213 AISTVGVT 221 DPRRGW 186 MA K DTRYCX3CR1BII308 140 GSIFSSNA 141 AINSVGVT 162 DPRRGW 186 MA K DTRY *CDRsequences were determined according to Antibody Engineering, vol 2 byKonetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Thesequence numbers in the table (SEQ) refer to the sequences in thesequence listing of the instant application.

In a further aspect, the present invention provides polypeptides havingone or more VHH domains.

In one aspect, a VHH domain of the present invention comprises oressentially consists of the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 3; or    -   b) amino acid sequences that have at least 90% amino acid        identity with the amino acid sequences of SEQ ID NO: 3; or    -   c) amino acid sequences that have 11, 10, 9, 8, 7, 6, 5, 4, 3,        2, or 1 amino acid difference with the amino acid sequences of        SEQ ID NO: 3 or    -   d) an amino acid sequence of any one of SEQ ID NO: 1-48, or SEQ        ID NO: 121-140, or SEQ ID NO: 222-224.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 49; or    -   b) an amino acid sequence that has at least 95% amino acid        identity with the amino acid sequences of SEQ ID NO: 49; or    -   c) an amino acid sequence that has 5, 4, 3, 2, or 1 amino acid        difference with the amino acid sequences of SEQ ID NO: 49; or    -   d) an amino acid sequence of any one of SEQ ID NO: 49-52.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 67; or    -   b) an amino acid sequence that has at least 90% amino acid        identity with the amino acid sequences of SEQ ID NO: 67; or    -   c) an amino acid sequence that has 12, 11, 10, 9, 8, 7, 6, 5, 4,        3, 2, or 1 amino acid difference with the amino acid sequences        of SEQ ID NO: 67; or    -   d) an amino acid sequence of any one of SEQ ID NO: 53-120.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the amino acid sequence set forth in any one ofSEQ ID NO: 121-140, or SEQ ID NO: 222-224.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the amino acid sequence set forth in any one ofSEQ ID NO: 138-140.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the amino acid sequence set forth in any one ofSEQ ID NO: 222-224.

Representative VHH domains of the present invention are shown in Table 5and representative optimized VHH domains of the present invention areshown in Table 6 below:

TABLE 5  VHH domains CX3CR1BIIPMP66B02EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 1AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP54A12EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 2AWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTI NO:SGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP54A3EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 3AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP54A4EVQLVESGRGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 4AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP54A5EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 5AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP54A7EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM SEQ ID 6AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTQVTVSS CX3CR1BIIPMP54B1EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 7AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTAYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP54B2EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM SEQ ID 8AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTLVTVSS CX3CR1BIIPMP54B3EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 9AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP54B5EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 10AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTAYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP54D5EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 11AWYRQAPPGKQRDLVALINSVGITKYADSVKGRFT NO:ISSDNAKNTVYLEMNSLKPEDTAVYYCTSDGRRG WDTRYWGQGTQVTVSS CX3CR1BIIPMP54D8EVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAM SEQ ID 12AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP54F6KVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 13AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP54G3EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 14AWYRQAPGKQRDLVALINSVGITKYADSVKGRFTI  NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP54H1EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM SEQ ID 15AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTV NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTQVTVSS CX3CR1BIIPMP54H4EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 16AWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTI NO:SGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP61F10KVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM SEQ ID 17AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTQVTVSS CX3CR1BIIPMP61D1EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 18AWYRQAFGKQRDLVALINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTQVTVSS CX3CR1BIIPMP61D5KVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 19AWYRQAFGKQRDLVALINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTQVTVSS CX3CR1BIIPMP61E2EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 20AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDMAVYYCTSDPRRG WDTRYWGQGTQVTVSS CX3CR1BIIPMP61F11KVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 21AWYROPPGKORDLVAAINSVGITKYADSVKGRFTI NO:FRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP61G2EVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 22AWYRQAPGKQRDLVALINSVGITKYADSVKGRFTI  NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP61G3KVQLVESGGGSMQAGESLRLSCAASGSIFSSNAM SEQ ID 23AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMMSLKPEDTAVYYCTSDPRRG WDTRYWGQGTQVTVSS CX3CR1BIIPMP61G4KVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAM SEQ ID 24AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMMSLKPEDTAVYYCTSDPRRG WDTRYWGQGTQVTVSS CX3CR1BIIPMP61F4EVQLVESGGGSVQAGASLRLSCAASGSIFSSNAM SEQ ID 25AWYRQAPGKQRDLVAVINTVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP61A11EVQLVESRGGSVQAGESLRLSCAASGSIFSSNAM  SEQ ID 26AWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTI NO:SGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP61B2EVQLVESRGGSVQAGASLRLSCAASGSIFSSNAM SEQ ID 27AWYRQAPGKQRDLVAVINTVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP61C9EVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 28AWYRQALGKQRDLVALIDSAGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNRLKPEDTAVYYCASDARRGW NTKYWGQGTLVTVSS CX3CR1BIIPMP65H02EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 29AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI NO:SRDNAKNTVHLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP65E11EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 30AWYRQAPGKQRDLVAGINSVGIAKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP65E10EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAKA SEQ ID 31WYRQAPGKQRDLVAGINSVGITKYADSVKGRFTIS NO:RDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTLVTVSS CX3CR1BIIPMP65E05KVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 32AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP65B11EVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 33AWYRQAPGKQRDLVAVINKVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP65B07EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 34AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP65B09EVQLVESGGGSVQAGESLRLSCAASGSIFSRNAM SEQ ID 35AWYRQAPGKQRDLVASINSVGITKYGDSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTLVTVSS CX3CR1BIIPMP65H01EVQLVESGGGSVQAGESLRLSCAASGGIFSRNAM SEQ ID 36AWYRQAPGKQRDLVASINSVGITKYGDSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP65G07EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM SEQ ID 37AWYRQAPGKQRDLVAGINSVDITRYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTQVTVSS CX3CR1BIIPMP66H08EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 38AWYRQAPGKQRDLVALINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP66H04EVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAM SEQ ID 39AWYRQAPGKQRDLVAAINSVGITKYADSVKGRFTI NO:SRDNAKNIVYLOMMSLKPEDTAVYYCTSDPRRG WDTRYWGQGTLVTVSS CX3CR1BIIPMP66F02EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 40AWYRQAPGKQRDLVALINSVGITKYAGSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP66E11EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 41AWYRQAPGKQRDLVAAINSVGTTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP66D10EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 42AWYRQALGKQRDLVALINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTQVTVSS CX3CR1BIIPMP66D08EVQLMESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 43AWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP66A04EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 44AWYRQALGKQRDLVALINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWD TRYWGQGTLVTVSS CX3CR1BIIPMP66D04KVQLVESGGGSVQAGESLRLSCAASGTIFSSNAM SEQ ID 45AWYRQAPGKQRDLVAGINSVDITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW NTRYWGQGTLVTVSS CX3CR1BIIPMP66D02EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 46AWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTT NO:SGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP66D06EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 47AWYRQAPGKQRDLVASIDSVGITKYRDSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGW DTRYWGQGTQVTVSS CX3CR1BIIPMP66G01EMQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 48AWYRQAPGKQRDLVALINSVGITKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDGRRG WDTRYWGQGTQVTVSS CX3CR1BIIPMP11H11EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAM SEQ ID 49GWFRQAPGKERAFVAGISGSASRKYYADSVKGRF NO:TVSRDNARNTVYLQMNSLKPEDTAVYYCAASNSY PKVQFDYYGQGTQVTVSS CX3CR1BIIPMP12B6EVQLVQSGGGLVQAGGSLRLSCVASGRTFSSYAM SEQ ID 50GWFRQAPGRERAFVAGISGSASRKYYADSVKGRF NO:TVSRDNARNTVYLQMNSLKPEDTAVYYCAASNSY PKVQFDYYGQGTQVTVSS CX3CR1BIIPMP12G9EVQLVESGGGLVQPGGSLRLSCVASGRTFSSYAM SEQ ID 51GWFRQAPGKEREFVAGISGSGSRKYYADSVKGRF NO:TISRDNARNTVYLQMNSLKPEDRAVYYCAASNSYP KVQFDYYGQGTQVTVSS CX3CR1BIIPMP15G11EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAM SEQ ID 52GWFRQAPGKEREFVAGISGSGSRKYYADSVKGRF NO:TISRDNARNTVYLQMNSLKPEDRAVYYCAASNSYP KVQFDYYGQGTQVTVSS CX3CR1BIIPMP18E6KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 53GWYRQAPGKKRDLVASISSSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRG WNTAYWGQGAQVTVSS CX3CR1BIIPMP12C2EVQLVESGGGLVQPGGSLRLSCATSGTIFSNTAM SEQ ID 54GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNSGYWGQGTQVTVSS CX3CR1BIIPMP18A10EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG SEQ ID 55WYRQAPGKKRDLVASISNSGSTNYADSAKGRFTV NO: SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP18A2 EVQLVESGGGVVQPGGSLRLSCVTSGIIFSNNAMGSEQ ID 56 WYRQGPGKKRDLVASIGSTYSTNYADSVKGRFTV NO:SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS CX3CR1BIIPMP18A8EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM SEQ ID 57GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP18A9EVQLVESGGGVVQPGGSLRLSCVTSGIIFSNNAMG SEQ ID 58WYRQGPGKKRDLVASISSTYSTNYADSVKGRFTV NO: SRDNDKNTGYLQMNSLKPEDTGVYYCTDARRGWNTAYWGQGTPVTVSS CX3CR1BIIPMP18B7 EVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAMSEQ ID 59 GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNSGYWGQGTQVTVSS CX3CR1BIIPMP18B9EVQLVESRGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 60GWYRQAPGKKRDLVASISSSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18C6EVQLMESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 61GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18C9EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG SEQ ID 62WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV NO: SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP18D1 EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGSEQ ID 63 WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV NO:SRDNDKSTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18D10 EVQLVESGGGLVQPGGSLGLSCATSGTIFSNNAM SEQ ID 64GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18D12EVQLVESGGGLVQPGGSLRLSCTTSGTIFSNNAM SEQ ID 65GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18F1KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 66GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18F5EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 67GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18F6EVQLVDSGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 68GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18F9EVQLVESGGGLVQPGGSLRLSCATSGTIFRTNAM SEQ ID 69GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP18G5EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM SEQ ID 70GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP18H1EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 71GWYRQALGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP18H10EVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAM SEQ ID 72GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP18H7EVQLVESRGGLVQPGGSLRLSCATSGIIFSNNAMG SEQ ID 73WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV NO: SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP18H9 EVQLVKSGGGLVQPGGSLRLSCTTSGTIFSNNAMSEQ ID 74 GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP20B3EVQLVESGGGLVQAGGSLRLSCVTSGIIFSNNAMG SEQ ID 75WYRQGPGKKRDLVASIGSTYSTNYADSVKGRFTV NO:SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS CX3CR1BIIPMP20C12EVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAM SEQ ID 76GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNSGYWGQGTRVTVSS CX3CR1BIIPMP20C3KVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG SEQ ID 77WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV NO: SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP20C6 EVQLVESGGGLVQAGGSLRLSCATSGTIFSNNAMSEQ ID 78 GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP20D8EVQLVESGGGLVQPGRSLRLSCATSGTTFRSNAM SEQ ID 79GWYRQGPGKKRDLVASITNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMSSLKPEDTGVYYCTLDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP20E11EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM SEQ ID 80GWYRQGPGKKRDLVASITNSGSTNYADSVKGRFT NO:VSRDNDRNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP20E5KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 81GWYRQVPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP20F3EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 82GWYRQAPGKKRDLVASISSSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP20F4EVQLVESGGGLVQPGGSLRLSCATSATIFRSNAM SEQ ID 83GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP20F5EVQLVESGGGLVQPGGSLRLSCATSATIFRSNAM SEQ ID 84GWYRQAPGKKRDLVASISNSGSTNYADSVKGRST NO:VSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP21B6 EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 85GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO: VSRDNDKNTGYLQMNSLKPEDMGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP24A12 EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGSEQ ID 86 WYRQAPGKKRDLVASISNSGSANYADSVKGRFTV NO:SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP24A6EVQLVESGGGLVQPGGSLRLSCTTSGTIFSNNAM SEQ ID 87GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSGDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP24B9EVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAM SEQ ID 88GWYRQAPGKKRDLVASISISGSTNYADSVKGRFTV NO:SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGFWGQGTQVTVSS CX3CR1BIIPMP24D3EVQLVESGGGLVQPGGSLRLSCVTSGIIFSNNAMG SEQ ID 89WYRQGPGKKRDLVASISSTYSTNYADSVKGRFTV NO:SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS CX3CR1BIIPMP24F7EVQLMESGGGMVQVGGSLRLSCTASGLIFSNNAM SEQ ID 90GWYRQGPGKKRDLVASISSTYSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTAYWGQGTPVTVSS CX3CR1BIIPMP28B4EVQLVESGGGLVQPGGSLRLSCAISATIFRSNAMG SEQ ID 91WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV NO:SRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRGW NTGYWGQGTQVTVSS CX3CR1BIIPMP28F1EMQLVESGGGVVQPGGSLRLSCVTSGIIFSNNAM  SEQ ID 92GWYRQGPGKKRDLVASIGSTYSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTAYWGQGTPVTVSS CX3CR1BIIPMP28F6EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG SEQ ID 93WYRQAPGKKRDLVASISNSGSTNHADSVKGRFTV NO: SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP28F9 EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMSEQ ID 94 GWYRQVPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP29A5EVQLVESRGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 95GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP29D5KVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAM SEQ ID 96GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNSGYWGQGTQVTVSS CX3CR1BIIPMP29E3EVQLVESEGGLVQPGGSLRLPCATSKTIFRSNAMG  SEQ ID 97WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV NO: SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIPMP29E7 EVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAMSEQ ID 98 GWYRQAPGKKRGLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP29G10EVQLMESGGGLVQPGGSLRLSCATSGTIFRSNAM SEQ ID 99GWYRQGPGKKRDLVASITNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMSSLKPEDTGVYYCTLDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP29G7EVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMG SEQ ID 100WYRQGPGKKRDLVASITNTGSTNYADSVKGRFTV NO: SRDNDRNTVYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP29H1 EVQLVESGGGLVQAGGSLRLSCTTSGTIFSNNAMSEQ ID 101 GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP37A8EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM SEQ ID 102GWYRQAPGKKRDLVASISNSGSTNYADSAKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP37B9EVQLVESGGLVQPGGSLRLSCATSGTIFSNNAMG SEQ ID 103WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV NO: SRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP37C12 EVQLVESGGGLVQAGGSLRLSCVASGSIFRSNAMSEQ ID 104 GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP37C7EVQLVESGGGLVQPGGSLRLSCATSRTIFSNNAM SEQ ID 105GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP37D9EVQLVESGGGLVQPGGSLRLSCATSGTVFSNNAM SEQ ID 106GWYRQAPGKKRDLVASISSSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRG WNTAYWGQGTQVTVSS CX3CR1BIIPMP37E12 EVQLVESGGGLVQPGGSLRLSCATSKPIFRSNAM SEQ ID 107GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP41B10 EVQLVESEGGLVQPGGSLRLSCTTSGTIFSNNAMG SEQ ID 108WYRQAPGKKRDLVASISNSGSTNYADSVKGRFTV NO: SRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIPMP41B11  EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMSEQ ID 109 GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSPKPEDTGVYYCTVDARR GWNTAYWGQGTQVTVSS CX3CR1BIIPMP41B8EVQLVESEGGVVQPGGSLRLSCVTSGIIFSNNAMG SEQ ID 110WYRQGPGKKRDLVASIGSTYSTNYADSVKGRFTV NO:SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS CX3CR1BIIPMP41C10EMQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM SEQ ID 111GRFTNDKSTGYLQMNSLKPEDTGVYYCTVDARRG NO:VSRDGWYRQAPGKKRDLVASISNSGSTNYADSVK WNTGYWGQGTQVTVSS CX3CR1BIIPMP41F9 EVQLVESGGGVVQPGESLRLSCVTSGIIFSNNAMG SEQ ID 112WYRQGPGKKRDLVASIGSTYSTNYADSVKGRFTV NO:SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS CX3CR1BIIPMP41H10KVQLVESGGGLVQPGDSLRLSCAASGLTLDDYAM SEQ ID 113GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP46B5KVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM SEQ ID 114GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP46D3EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 115GWYRQVPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLRMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP46H5 EVQLVESGGGLVQAGGSLRLSCVTSGIIFSNNAMG SEQ ID 116WYRQGPGKKRDLVASISSTYSTNYADSVKGRFTV NO:SRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGW NTAYWGQGTPVTVSS CX3CR1BIIPMP48B8EVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAM SEQ ID 117GWYRQAPGKKRDLVASISNSGSTNYTDSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP48D11KVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM SEQ ID 118GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP48G8EVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAM SEQ ID 119GWYRQAPGKKRDLVASISNSGSTNYADSVKGRFA NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTGYWGQGTQVTVSS CX3CR1BIIPMP48H9EVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAM SEQ ID 120GWYRQAPGKKRDLVASISNSGSTNYADFVKGRFT NO:VSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRG WNTAYWGQGTQVTVSS SEQ ID NO: 1-48 areVHH domains of family 101. SEQ ID NO: 49-52 are VHH domains of family 9.SEQ ID NO: 53-120 are VHH domains of family 13.

TABLE 6  Optimized VHH domains CX3CR1BII043EVQLVESGGGSVQPGESLRLSCAASGSIFSSNAM SEQ ID 121AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII045DVQLVESGGGSVQPGESLRLSCAASGSIFSSNAM SEQ ID 122AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII047EVQLVESGGGLVQPGESLRLSCAASGSIFSSNAMA SEQ ID 123WYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTIS NO:RDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII048EVQLVESGGGSVQPGGSLRLSCAASGSIFSSNAM SEQ ID 124AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII049EVQLVESGGGSVQPGESLRLSCAASGSIFSSNAM SEQ ID 125AWYRQAPGKQRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII050EVQLVESGGGSVQPGESLRLSCAASGSIFSSNAM SEQ ID 126AWYRQAPGKRRELVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII061EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM SEQ ID 127AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII056EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM SEQ ID 128AWYRQAPGKQRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII057EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM SEQ ID 129AWYRQAPGKRRELVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII060EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM SEQ ID 130AWYRQAPGKQRELVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII065EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 131AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII067EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 132AWYRQAPGKRRDLVAAIQSVGVTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII068EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 133AWYRQAPGKRRDLVAAIGSVGVTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII074EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 134AWYRQAPGKRRDLVAAITSVGVTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII118EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 135AWYRQAPGKRRDLVAAINTVGVTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII129EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 136AWYRQAPGKRRDLVAAINGVGVTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII158EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAM SEQ ID 137AWYRQAPGKRRDLVAAINPVGVTKYADSVKGRFTI NO:SRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII306DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ ID 138AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII307DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ ID 139AWYRQAPGKRRDLVAAISTVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII308DVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM SEQ ID 140AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII00306EVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMA SEQ ID 222 (D1E)WYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTIS NO:RDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII00307EVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMA SEQ ID 223 (D1E)WYRQAPGKRRDLVAAISTVGVTKYADSVKGRFTIS NO:RDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS CX3CR1BII00308EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM SEQ ID 224 (D1E)AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTI NO:SRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGW DTRYWGQGTLVTVSS

In a further aspect, a polypeptide according to the present invention,in particular an immunoglobulin single variable domain of the presentinvention, is humanized and/or optimized for stability, potency,manufacturability and/or similarity to human framework regions. Forexample, the polypeptide is humanized and/or sequence optimized in oneor more of the following positions (according to Kabat numbering): 1,11, 14, 16, 74, 83, 108. In one aspect, the polypeptide comprises one ormore of the following mutations: E1D, S11 L, A14P, E16G, A74S, K83R,Q108L.

In one aspect, one or more framework regions of a polypeptide accordingto the present invention, in particular an immunoglobulin singlevariable domain of the present invention, are humanized and/or sequenceoptimized. In one aspect, a polypeptide according to the presentinvention, in particular an immunoglobulin single variable domain of thepresent invention, comprises framework regions (FR) for example as setforth below:

-   -   i) FR1 is selected from any one of SEQ ID NO's: 198-204;    -   ii) FR2 is selected from any one of SEQ ID NO's: 205-208;    -   iii) FR3 is selected form any one of SEQ ID NO's: 209-210;        and/or    -   iv) FR4 is selected from any one of SEQ ID NO's: 211-212.

Human immunoglobulin framework region sequences (FR) that can also beused as framework region sequences for the immunoglobulin singlevariable domains as described above are known in the art. Also known inthe art are methods for humanizing framework regions of immunoglobulinsingle variable domains derived from species other than humans.

In a further aspect, one or more CDR regions of a polypeptide accordingto the present invention, in particular an immunoglobulin singlevariable domain of the present invention, is humanized and/or sequenceoptimized. In one aspect, a polypeptide according to the presentinvention, in particular an immunoglobulin single variable domain of thepresent invention, is humanized and/or sequence optimized in one or moreof the following positions (according to Kabat numbering): 52, 53.

In a further aspect, a polypeptide according to the present invention,in particular an immunoglobulin single variable domain of the presentinvention, comprises one or more of the following mutations: N52S, S53T.

In a further aspect, a polypeptide according to the present invention,in particular an immunoglobulin single variable domain of the presentinvention, comprises a CDR2 selected from any one of SEQ ID NO's:214-221.

Representative humanized and/or optimized sequences of the presentinvention are shown in Table 4 and 6 hereinabove and in Table 7 hereinbelow.

Table 7: Sequence Optimized Variants

Table 7a shows the FR1-CDR1-FR2-CRD2 of the sequence optimized variants,table 7b shows FR3-CDR3-FR4-CDR4 of said variants. The sequence numbersin the tables (SEQ) refer to the sequences in the sequence listing ofthe instant application.

TABLE 7a  Sequence optimized variants (FR1-CDR1-FR2-CDR2) SEQ SEQ SEQSEQ Nanobody SEQ FR1 FR1 CDR1 CDR1 FR2 FR2 CDR2 CDR2 CX3CR1BIIPMP66B02  1 EVQLVES 198 GSIFS 141 WYRQ 205 AINSV 162 GGGSVQ SNAM APGKR GVTKAGESLRL A RDLVA SCAAS CX3CR1BII043 121 EVQLVES 199 GSIFS 141 WYRQ 205AINSV 162 GGGSVQ SNAM APGKR GVTK PGESLRL A RDLVA SCAAS CX3CR1BII045 122DVQLVES 200 GSIFS 141 WYRQ 205 AINSV 162 GGGSVQ SNAM APGKR GVTK PGESLRLA RDLVA SCAAS CX3CR1BII047 123 EVQLVES 201 GSIFS 141 WYRQ 205 AINSV 162GGGLVQ SNAM APGKR GVTK PGESLRL A RDLVA SCAAS CX3CR1BII048 124 EVQLVES202 GSIFS 141 WYRQ 205 AINSV 162 GGGSVQ SNAM APGKR GVTK PGGSLRL A RDLVASCAAS CX3CR1BII049 125 EVQLVES 199 GSIFS 141 WYRQ 206 AINSV 162 GGGSVQSNAM APGK GVTK PGESLRL A QRDLV SCAAS A CX3CR1BII050 126 EVQLVES 199GSIFS 141 WYRQ 207 AINSV 162 GGGSVQ SNAM APGKR GVTK PGESLRL A RELVASCAAS CX3CR1BII061 127 EVQLVES 203 GSIFS 141 WYRQ 205 AINSV 162 GGGLVQSNAM APGKR GVTK PGGSLRL A RDLVA SCAAS CX3CR1BII056 128 EVQLVES 203 GSIFS141 WYRQ 206 AINSV 162 GGGLVQ SNAM APGK GVTK PGGSLRL A QRDLV SCAAS ACX3CR1BII057 129 EVQLVES 203 GSIFS 141 WYRQ 207 AINSV 162 GGGLVQ SNAMAPGKR GVTK PGGSLRL A RELVA SCAAS CX3CR1BII060 130 EVQLVES 203 GSIFS 141WYRQ 208 AINSV 162 GGGLVQ SNAM APGK GVTK PGGSLRL A QRELV SCAAS ACX3CR1BII065 131 EVQLVES 198 GSIFS 141 WYRQ 205 AISSV 214 GGGSVQ SNAMAPGKR GVTK AGESLRL A RDLVA SCAAS CX3CR1BII067 132 EVQLVES 198 GSIFS 141WYRQ 205 AIQSV 215 GGGSVQ SNAM APGKR GVTK AGESLRL A RDLVA SCAASCX3CR1BII068 133 EVQLVES 198 GSIFS 141 WYRQ 205 AIGSV 216 GGGSVQ SNAMAPGKR GVTK AGESLRL A RDLVA SCAAS CX3CR1BII074 134 EVQLVES 198 GSIFS 141WYRQ 205 AITSV 217 GGGSVQ SNAM APGKR GVTK AGESLRL A RDLVA SCAASCX3CR1BII118 135 EVQLVES 198 GSIFS 141 WYRQ 205 AINTV 218 GGGSVQ SNAMAPGKR GVTK AGESLRL A RDLVA SCAAS CX3CR1BII129 136 EVQLVES 198 GSIFS 141WYRQ 205 AING 219 GGGSVQ SNAM APGKR VGVT AGESLRL A RDLVA K SCAASCX3CR1BII158 137 EVQLVES 198 GSIFS 141 WYRQ 205 AINPV 220 GGGSVQ SNAMAPGKR GVTK AGESLRL A RDLVA SCAAS CX3CR1BII306 138 DVQLVES 204 GSIFS 213WYRQ 205 AISSV 214 GGGLVQ STAMA APGKR GVTK PGGSLRL RDLVA SCAASCX3CR1BII307 139 DVQLVES 204 GSIFS 213 WYRQ 205 AISTV 221 GGGLVQ STAMAAPGKR GVTK PGGSLRL RDLVA SCAAS CX3CR1BII308 140 DVQLVES 204 GSIFS 141WYRQ 205 AINSV 162 GGGLVQ SNAM APGKR GVTK PGGSLRL A RDLVA SCAAS

TABLE 7b  Sequence optimized variants (FR3-CDR3-FR4) Nano SEQ SEQ SEQbody SEQ FR3 FR3 CDR3 CDR3 FR4 FR4 CX3CR1BIIPMP66B02 1 YADSVKGRFTI 209DPRRGW 186 WGQGTQ 211 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTSCX3CR1BII043 121 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 SRDNSKNTVYL DTRYVTVSS QMNSLRPEDT AVYYCTS CX3CR1BII045 122 YADSVKGRFTI 210 DPRRGW 186WGQGTL 212 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1BII047 123YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDTAVYYCTS CX3CR1BII048 124 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1BII049 125 YADSVKGRFTI210 DPRRGW 186 WGQGTL 212 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTSCX3CR1BII050 126 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 SRDNSKNTVYL DTRYVTVSS QMNSLRPEDT AVYYCTS CX3CR1BII061 127 YADSVKGRFTI 210 DPRRGW 186WGQGTL 212 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1BII056 128YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDTAVYYCTS CX3CR1BII057 129 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1BII060 130 YADSVKGRFTI210 DPRRGW 186 WGQGTL 212 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTSCX3CR1BII065 131 YADSVKGRFTI 209 DPRRGW 186 WGQGTL 212 SRDNAKNTVYL DTRYVTVSS QMNSLKPEDTA VYYCTS CX3CR1BII067 132 YADSVKGRFTI 209 DPRRGW 186WGQGTL 212 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTS CX3CR1BII068 133YADSVKGRFTI 209 DPRRGW 186 WGQGTL 212 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTAVYYCTS CX3CR1BII074 134 YADSVKGRFTI 209 DPRRGW 186 WGQGTL 212SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTS CX3CR1BII118 135 YADSVKGRFTI209 DPRRGW 186 WGQGTL 212 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTSCX3CR1BII129 136 YADSVKGRFTI 209 DPRRGW 186 WGQGTL 212 SRDNAKNTVYL DTRYVTVSS QMNSLKPEDTA VYYCTS CX3CR1BII158 137 YADSVKGRFTI 209 DPRRGW 186WGQGTL 212 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTS CX3CR1BII306 138YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDTAVYYCTS CX3CR1BII307 139 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1BII308 140 YADSVKGRFTI210 DPRRGW 186 WGQGTL 212 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS

In one aspect of the present invention, a polypeptide of the inventioncan additionally contain modifications such as glycosyl residues,modified amino acid side chains, and the like.

It will be clear to the skilled person that for pharmaceutical uses inhumans, the polypeptides of the invention are preferably directedagainst human CX3CR1, whereas for veterinary purposes, the polypeptidesof the invention are preferably directed against CX3CR1 from the speciesto be treated.

It will also be clear to the skilled person that when used as atherapeutic agent in humans, the immunoglobulin single variable domainscomprised in the polypeptides according to the invention are preferablyhumanized immunoglobulin single variable domains.

According to the invention, an immunoglobulin single variable domain canbe a domain antibody, i.e. VL or VH antibody, and/or VHH domains asdescribed above, and/or any other sort of immunoglobulin single variabledomain, for example camelized VH, provided that these immunoglobulinsingle variable domains are anti-CX3CR1 immunoglobulin single variabledomains.

In one aspect of the invention, the immunoglobulin single variabledomain essentially consists of either a domain antibody sequence or aVHH domain sequence as described above. In particular, theimmunoglobulin single variable domain essentially consists of a VHHdomain sequences.

In a further aspect, a polypeptide of the present invention comprisestwo or more anti-CX3CR1 immunoglobulin single variable domains. In afurther aspect, a polypeptide of the present invention comprises twoanti-CX3CR1 immunoglobulin single variable domains, for exampleanti-CX3CR1 VHHs. In one aspect, the two anti-CX3CR1 immunoglobulinsingle variable domains in a polypeptide of the present invention havethe same amino acid sequence. In another aspect, the two anti-CX3CR1immunoglobulin single variable domains in a polypeptide of the presentinvention have different amino acid sequences.

According to another embodiment of the invention, the at least twoimmunoglobulin single variable domains present in a polypeptide of theinvention can be linked to each other directly (i.e. without use of alinker) or via a linker. The linker is preferably a linker peptide andwill, according to the invention, be selected so as to allow binding ofthe at least two immunoglobulin single variable domains to CX3CR1,either within one and the same CX3CR1 molecule, or within two differentmolecules.

Suitable linkers will inter alia depend on the epitopes and,specifically, the distance between the epitopes on CX3CR1 to which theimmunoglobulin single variable domains bind, and will be clear to theskilled person based on the disclosure herein, optionally after somelimited degree of routine experimentation.

Also, when the two or more anti-CX3CR1 immunoglobulin single variabledomains are domain antibodies or VHH domains, they may also be linked toeach other via a third domain antibody or VHH domain (in which the twoor more immunoglobulin single variable domains may be linked directly tothe third domain antibody or VHH domain or via suitable linkers). Such athird domain antibody or VHH domain may for example be a domain antibodyor VHH domain that provides for an increased half-life, as furtherdescribed herein. For example, the latter domain antibody or VHH domainmay be a domain antibody or VHH domain that is capable of binding to a(human) serum protein such as (human) serum albumin or (human)transferrin, as further described herein.

Alternatively, the two or more anti-CX3CR1 immunoglobulin singlevariable domains may be linked in series (either directly or via asuitable linker) and the third (single) domain antibody or VHH domain(which may provide for increased half-life, as decribed above) may beconnected directly or via a linker to one of these two or moreaforementioned immunoglobulin sequences.

Suitable linkers are described herein in connection with specificpolypeptides of the invention and may—for example and withoutlimitation—comprise an amino acid sequence, which amino acid sequencepreferably has a length of 5 or more amino acids, 7 or more amino acids,9 or more amino acids, 11 or more amino acids, 15 or more amino acids orat least 17 amino acids, such as about 20 to 40 amino acids. However,the upper limit is not critical but is chosen for reasons of convenienceregarding e.g. biopharmaceutical production of such polypeptides.

The linker sequence may be a naturally occurring sequence or anon-naturally occurring sequence. If used for therapeutical purposes,the linker is preferably non-immunogenic in the subject to which thepolypeptide of the invention is administered.

One useful group of linker sequences are linkers derived from the hingeregion of heavy chain antibodies as described in WO 96/34103 and WO94/04678.

Other examples are poly-alanine linker sequences such as Ala-Ala-Ala.

Further preferred examples of linker sequences are Gly/Ser linkers ofdifferent length such as (gly_(x)ser_(y)), linkers, including(gly₄ser)₃, (gly₄ser)₄, (gly₄ser), (gly₃ser), gly₃, and (gly₃ser₂)₃.

If the polypeptide of the invention is modified by the attachment of apolymer, for example a polyethylene glycol (PEG) moiety, the linkersequence preferably includes an amino acid residue, such as a cysteineor a lysine, allowing such modification, e.g. PEGylation, in the linkerregion.

Examples of linkers are:

(5 GS linker, SEQ ID NO: 233) GGGGS (7GS linker, SEQ ID NO: 234) SGGSGGS(8GS linker, SEQ ID NO: 235) GGGGCGGGS (9GS linker, SEQ ID NO: 236)GGGGSGGGS (10GS linker, SEQ ID NO: 237) GGGGSGGGGS(15GS linker, SEQ ID NO: 238) GGGGSGGGGSGGGGS(18GS linker, SEQ ID NO: 239) GGGGSGGGGSGGGGGGGS(20GS linker, SEQ ID NO: 240) GGGGSGGGGSGGGGSGGGGS(25GS linker, SEQ ID NO: 241) GGGGSGGGGSGGGGSGGGGSGGGGS(30GS linker, SEQ ID NO: 242) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(35GS linker, SEQ ID NO: 243) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(G1 hinge linker, SEQ ID NO: 244) EPKSCDKTHTCPPCP(9GS-G1 hinge linker, SEQ ID NO: 245) GGGGSGGGSEPKSCDKTHTCPPCP(Llama upper long hinge region, SEQ ID NO: 246) EPKTPKPQPAAA(G3 hinge, SEQ ID NO: 247)ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPK SCDTPPPCPRCP(Ala linker, SEQ ID NO: 248) AAA 

Furthermore, the linker may also be a poly(ethylene glycol) moiety, asshown in e.g. WO04/081026.

Non-limiting examples of polypeptides comprising or consisting of two ormore anti-CX3CR1 immunoglobulin single variable domains are given inTable 8a.

TABLE 8a  Bivalent anti-CX3CR1 polypeptides CX3CR1BII007EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG SEQ 267WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV IDSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV NO:QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSKVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASIS SSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNTAYWGQGAQVTVSS CX3CR1BII009KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMG SEQ  268WYRQAPGKKRDLVASISSSGSTNYADSVKGRFTVS IDRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNT NO: AYWGQGAQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCVASGRTFSSYAMGWFRQAPGKERAFVAGISGSASRKYYADSVKGRFTVSRDNARNTVYLQMNSLK PEDTAVYYCAASNSYPKVQFDYYGQGTQVTVSSCX3CR1BII012 EVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAMA SEQ  269WYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISR IDDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR NO:YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSKVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASISSSGS TNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNTAYWGQGAQVTVSS CX3CR1BII016EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ  270WYRQAPGKQRDLVAVINSVGITKYADSVKGRFTISG IDDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTR NO: YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTISGDNAKNTVYLQMNSLKPEDT AVYYCTSDARRGWDTRYWGQGTQVTVSSCX3CR1BII017 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ  271WYRQAPPGKQRDLVALINSVGITKYADSVKGRFTISS IDDNAKNTVYLEMNSLKPEDTAVYYCTSDGRRGWDTR NO: YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPPGKQRDLVALINSVGITKYADSVKGRFTISSDNAKNTVYLEMNSLKPEDT AVYYCTSDGRRGWDTRYWGQGTQVTVSSCX3CR1BII018 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ  272WYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR IDDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR NO: YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCTSDPRRGWDTRYWGQGTQVTVSSCX3CR1BII019 EMQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ  273WYRQAPGKQRDLVALINSVGITKYADSVKGRFTI SR IDDNAKNTVYLQMNSLKPEDTAVYYCTSDGRRGWDTR NO: YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEMQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKQRDLVALINSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCTSDGRRGWDTRYWGQGTQVTVSSCX3CR1BII020 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ  274WYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISR IDDNAKNTAYLQMNSLKPEDTAVYYCTSDPRRGWDTR NO:YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAV YYCTSDPRRGWDTRYWGQGTLVTVSSCX3CR1BII026 EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG SEQ  275WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV IDSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV NO:QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCTSDPRRGWDTRYWGQGTQVTVSSCX3CR1BII027 EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG SEQ  276WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV IDSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV NO:QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISRDNAKNTAYLQMNSLKPE DTAVYYCTSDPRRGWDTRYWGQGTLVTVSSCX3CR1BII006 EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG SEQ  282WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV IDSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV NO:QFDYYGQGTLVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMGWFRQAPGKERAFVAGI SGSASRKYYADSVKGRFTVSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKVQFDYYGQGTLVTVSS

In another embodiment, the at least two immunoglobulin single variabledomains of the polypeptide of the invention are linked to each other viaanother moiety (optionally via one or two linkers), such as anotherpolypeptide which, in a preferred but non-limiting embodiment, may be afurther immunoglobulin single variable domain as already describedabove. Such moiety may either be essentially inactive or may have abiological effect such as improving the desired properties of thepolypeptide or may confer one or more additional desired properties tothe polypeptide. For example, and without limitation, the moiety mayimprove the half-life of the protein or polypeptide, and/or may reduceits immunogenicity or improve any other desired property.

In one aspect, a polypeptide of the invention includes, especially whenused as a therapeutic agent, a moiety which extends the half-life of thepolypeptide of the invention in serum or other body fluids of a patient.The term “half-life” means the time taken for the serum concentration ofthe (modified) polypeptide to reduce by 50%, in vivo, for example due todegradation of the polypeptide and/or clearance and/or sequestration bynatural mechanisms.

According to a further embodiment of the invention, the twoimmunoglobulin single variable domains may be fused to a serum albuminmolecule, such as described e.g. in WO01/79271 and WO03/59934.

Alternatively, such half-life extending moiety can be covalently linkedor fused to said polypeptide and may be, without limitation, an Fcportion, an albumin moiety, a fragment of an albumin moiety, an albuminbinding moiety, such as an anti-albumin immunoglobulin single variabledomain, a transferrin binding moiety, such as an anti-transferrinimmunoglobulin single variable domain, a polyoxyalkylene molecule, suchas a polyethylene glycol molecule, an albumin binding peptide, orhydroxyethyl starch (HES) derivatives.

In another aspect, the polypeptide of the invention comprises a moietywhich binds to an antigen found in blood, such as serum albumin, serumimmunoglobulins, thyroxine-binding protein, fibrinogen or transferrin,thereby conferring an increased half-life in vivo to the resultingpolypeptide of the invention. According to one embodiment, such moietyis an albumin-binding immunoglobulin and, in particular, analbumin-binding immunoglobulin single variable domain such as analbumin-binding VHH domain.

In another embodiment, the polypeptide of the invention comprises amoiety which binds to serum albumin, wherein such moiety is an albuminbinding peptide, as described e.g. in international patent publicationsWO2008/068280 and WO2009/127691.

If intended for use in humans, such albumin-binding immunoglobulinsingle variable domain (also called anti-albumin immunoglobulin singlevariable domain) will preferably bind to human serum albumin and willpreferably be a humanized albumin-binding VHH domain.

Immunoglobulin single variable domains binding to human serum albuminare known in the art and are described in further detail in e.g.WO2006/122786. A specifically useful albumin binding VHH domain consistsof or contains the amino acid sequence as set forth in any one of SEQ IDNO: 230-232:

TABLE 8b  ALB-1 AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFG SEQ 230MSWVRQAPGKEPEWVSSISGSGSDTLYADSVK ID GRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGNO: GSLSRSSQGTQVTVSS ALB-11 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFG SEQ 231(humanized  MSWVRQAPGKGLEWVSSISGSGSDTLYADSVK ID ALB-1)GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG NO: GSLSRSSQGTLVTVSS ALB-2AVQLVESGGGLVQGGGSLRLACAASERIFDLNL SEQ 232MGWYRQGPGNERELVATCITVGDSTNYADSVK ID GRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIRNO: RTWHSELWGQGTQVTVSS

According to one embodiment, a polypeptide of the invention may belinked to one or more antibody parts, fragments or domains that conferone or more effector functions to the polypeptide of the inventionand/or may confer the ability to bind to one or more Fc receptors. Forexample, for this purpose, and without being limited thereto, theantibody parts may be or may comprise CH2 and/or CH3 domains of anantibody, such as from a heavy chain antibody (as described hereabove)and more preferably from a conventional human 4-chain antibody;specifically, the polypeptide of the invention may be linked to an Fcregion, for example from human IgG, from human IgE or from another humanIg. For example, WO 94/04678 describes heavy chain antibodies comprisinga Camelid VHH domain or a humanized derivative thereof, in which theCamelidae CH2 and/or CH3 domain have been replaced by human CH2 and/orCH3 domains, so as to provide an immunoglobulin that consists of 2 heavychains each comprising a—optionally humanized—VHH domain and human CH2and CH3 domains (but no CH1 domain), which immunoglobulin has theeffector function provided by the CH2 and CH3 domains, can functionwithout the presence of any light chains, and has an increased half-lifeas compared to the corresponding VHH domains without such modification.

In one aspect, a polypeptide of the present invention comprises twoanti-CX3CR1 VHHs and a VHH capable of binding to serum albumin. In oneaspect, the VHHs are fused using linker peptides. Representativeexamples of such polypeptides of the present invention are shownhereinbelow.

In one aspect, a polypeptide of the present invention comprises a firstanti-CX3CR1 VHH fused to a first linker peptide, which is itself fusedto a VHH capable of binding to serum albumin, which is itself fused to asecond linker peptide, which is itself fused to a second anti-CX3CR1VHH. In one aspect, the first or the second linker peptide is a 9GSlinker, in one aspect, the first and the second linker peptide is a 9GSlinker. In one aspect, the VHH capable of binding to serum albumin iscapable of binding to human serum albumin. In one aspect, the VHHcapable of binding to serum albumin has the amino acid sequence setforth in SEQ ID NO: 231. In one aspect, the first and the secondanti-CX3CR1 VHH have the same amino acid sequence. In one aspect, thefirst or the second anti-CX3CR1 VHH has the CDR1, CDR2 and CDR3 setforth in:

-   -   SEQ ID NO's: 213, 214 and 186; or    -   SEQ ID NO's: 213, 221 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

In one aspect, the first and the second anti-CX3CR1 VHH have the CDR1,CDR2 and CDR3 set forth in:

-   -   SEQ ID NO's: 213, 214 and 186; or    -   SEQ ID NO's: 213, 221 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

In one aspect, the first or the second anti-CX3CR1 VHH has the aminoacid sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ IDNO: 222 to 224. In one aspect, the first and the second anti-CX3CR1 VHHhave the same amino acid sequence, wherein said amino acid sequence isthe sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ ID NO:222 to 224.

Non-limiting examples of polypeptides of the present invention are thepolypeptides of any one of SEQ ID NO: 225 to 227, 249 or 277 to 281.

TABLE 9 CX3CR1BII00312 DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ ID 225MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR NO: FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BII00313 DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ ID 226MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR NO: FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLVAAISTVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BII00314 DVQLVESGGGLVQPGGSLRLSCAASGSIFSSNA SEQ ID 227MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR NO: FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNSKNTV YLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BII032 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ ID 277MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR NO: FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGSEVQL VESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGSEVQLVES GGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS CX3CR1BII034 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ ID 278MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR NO: FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQ APGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPG KGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTL VTVSS CX3CR1BII036EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ ID 249MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR NO: FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGSV QAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVY LQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BII040 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ ID 279MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR NO: FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGSEVQL VESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGK GLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS CX3CR1BII041EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ ID 280MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR NO: FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQ APGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQP GNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLY LQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVS SCX3CR1BII042 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ ID 281MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR NO: FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRL SCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKP EDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS

In another aspect, a polypeptide of the present invention comprises ananti-CX3CR1 VHH and a Fc domain. In one aspect, a polypeptide of thepresent invention comprises an anti-CX3CR1 VHH fused to a linkerpeptide, which is itself fused to a Fc domain. In one aspect, the linkerpeptide is a 15GS linker. In one aspect, the Fc domain has the aminoacid sequence set forth in SEQ ID NO: 250 or 252. In one aspect, the VHHhas the CDR1, CDR2 and CDR3 set forth in:

-   -   SEQ ID NO's: 213, 214 and 186; or    -   SEQ ID NO's: 213, 221 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

In one aspect, the VHH has the amino acid sequence set forth in any oneof SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224. In one aspect thepolypeptide is in the form of a dimer, for example wherein the dimer isformed by one or more disulfide bridge.

Non-limiting examples of polypeptides of the present invention are thepolypeptides of SEQ ID NO: 251, 253 or 254.

TABLE 10  Mouse Fc PPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIV SEQ  250 domainTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHR ID EDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKNO: DLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTK KQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS VVHEGLHNHHTTKSFSRTPGK 66602-EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ  251 mFcMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVICVVVAVSEDDPDVQ1SWFVNNVEVHTAQTQ THREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSY SCSVVHEGLHNHHTTKSFSRTPGK Human FcCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV SEQ  252 DomainTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR ID EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNNO: KALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 306D-hFcDVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ  253MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 307D-hFcDVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ  254MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

A polypeptide of the invention may be modified to improve itsproperties. In one aspect, a polypeptide of the present invention may bemodified to increase its stability upon storage. In one aspect, apolypeptide of the present invention may be modified to facilitate itsexpression in a particular host system. For example, the first codon ofa polypeptide of the present invention may be modified. In one aspect, apolypeptide of the present invention begins with a glutamic acid (glu)as its first amino acid. In another aspect, a polypeptide of the presentinvention begins with an aspartic acid (asp) as its first amino acid,for example to reduce pyroglutamate formation at the N-terminus duringstorage and hence increase product stability. In another aspect, apolypeptide of the present invention begins with an alanine (ala) or avaline (val) as its first amino acid, for example to facilitate theexpression of the polypeptide in a prokaryotic expression system, suchas Escherichia coli. Such modification of a polypeptide according to thepresent invention are made using techniques known in the art.

Representative examples of polypeptides according to the presentinvention with a modified first codon are set forth in any one of SEQ IDNO: 257-262 and 263-266 and are shown in Tables 11 and 12 below:

TABLE 11  CX3CR1BII00312 AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ ID 257(D1A) MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR NO:FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRR DLVAAISSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS CX3CR1BII00313AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ ID 258 (D1A)MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR NO: FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLVAAISTVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BII00314 AVQLVESGGGLVQPGGSLRLSCAASGSIFSSNA SEQ ID 259(D1A) MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR NO:FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKR RDLVAAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQ GTLVTVSS CX3CR1BII00312VQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ ID 260 (D1V)AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRF NO: TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSSGGGGSGGGSEVQLV ESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQ PGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BII00313 VQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ ID 261(D1V) AWYRQAPGKRRDLVAAISTVGVTKYADSVKGRF NO:TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR GWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRD LVAAISTVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTL VTVSS CX3CR1BII00314VQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM SEQ ID 262 (D1V)AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRF NO: TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSSGGGGSGGGSEVQLV ESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQ PGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS

TABLE 12  306D-hFc AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 263 (D1A)MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 307D-hFcAVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 264 (D1A)MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 306D-hFcVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ 265 (D1V)AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRF ID TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRNO: GWDTRYWGQGTLVTVSSGGGGSGGGGSGGG GSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 307D-hFcVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ 266 (D1V)AWYRQAPGKRRDLVAAISTVGVTKYADSVKGRF ID TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRNO: GWDTRYWGQGTLVTVSSGGGGSGGGGSGGG GSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

In one further aspect, a polypeptide of the present invention ischaracterized by one or more of the following properties:

-   -   Bind with high affinity to human CX3CR1;    -   Inhibit binding of soluble fractalkine to human CX3CR1;    -   Inhibit fractalkine induced chemotaxis;    -   Inhibit fractalkine induced human CX3CR1 receptor        internalization;    -   Cross-react with cyno CX3CR1 within 10-fold of E/IC₅₀ for human        CX3CR1 for binding and functional inhibition.

Accordingly, in one aspect, a polypeptide of the present invention hasan affinity to human CX3CR1 at an IC50 less than or equal to 10 nM, orless than or equal to 5 nM, or less than or equal to 2.5 nM or less thanor equal to 1 nM, as determined by competition FACS.

In a further aspect, a polypeptide of the present invention has anaffinity to human CX3CR1 at an EC50 of less than or equal to 10 nM, orless than or equal to 5 nM, or less than or equal to 2.5 nM or less thanor equal to 1 nM, as determined by cell binding FACS.

In a further aspect, a polypeptide of the present invention blocks thebinding of human CX3CR1 to human fractalkine at or above 50%, or at orabove 60%, or at or above 70%, or at or above 80%, or at or above 90%,or at or above 95% as determined by competition FACS with humanfractalkine.

In a further aspect, a polypeptide of the present invention blocks thebinding of human fractalkine to human CX3CR1 at an 1050 of less than orequal to 300 nM, or less than or equal to 100 nM, or less than or equalto 20 nM, or less than or equal to 10 nM, less than or equal to 5 nM,less than or equal to 2.5 nM or less than or equal to 1 nM as determinedby competition FACS with human fractalkine.

In a further aspect, a polypeptide of the present invention inhibitsfractalkine induced chemotaxis mediated by human CX3CR1 at or above 10%,or at or above 30%, or at or above 40%, or at or above 50%, or at orabove 60%, or at or above 70%, or at or above 80%, or at or above 90%.

In a further aspect, a polypeptide of the present invention inhibitsfractalkine induced chemotaxis mediated by human CX3CR1 at an IC₅₀ ofless than or equal to 500 nM, or of less than or equal to 100 nM, orless than or equal to 75 nM, or less than or equal to 50 nM, or lessthan or equal to 10 nM or less than or equal to 5 nM.

In a further aspect, a polypeptide of the present invention inhibitsfractalkine induced human CX3CR1 receptor internalization at an IC₅₀ ofless than or equal to 10 nM, or less than or equal to 5 nM or or lessthan or equal to 1 nM.

According to still another embodiment, a half-life extendingmodification of a polypeptide of the invention (such modification alsoreducing immunogenicity of the polypeptide) comprises attachment of asuitable pharmacologically acceptable polymer, such as straight orbranched chain poly(ethylene glycol) (PEG) or derivatives thereof (suchas methoxypoly(ethylene glycol) or mPEG). Generally, any suitable formof PEGylation can be used, such as the PEGylation used in the art forantibodies and antibody fragments (including but not limited to domainantibodies and scFv's); reference is made, for example, to: Chapman,Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug.Discov. 2 (2003); WO 04/060965; and U.S. Pat. No. 6,875,841.

Various reagents for PEGylation of polypeptides are also commerciallyavailable, for example from Nektar Therapeutics, USA, or NOFCorporation, Japan, such as the Sunbright® EA Series, SH Series, MASeries, CA Series, and ME Series, such as Sunbright® ME-100MA,Sunbright® ME-200MA, and Sunbright® ME-400MA.

Preferably, site-directed PEGylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering 16,761-770 (2003)). For example, for this purpose, PEG may be attached to acysteine residue that naturally occurs in a polypeptide of theinvention, a polypeptide of the invention may be modified so as tosuitably introduce one or more cysteine residues for attachment of PEG,or an amino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus and/or PEGmay be attached to a linker region that bridges two or more functionaldomains of a polypeptide of the invention, all using techniques ofprotein engineering known per se to the skilled person.

Preferably, for the polypeptides of the invention, a PEG is used with amolecular weight of more than 5 kDa, such as more than 10 kDa and lessthan 200 kDa, such as less than 100 kDa; for example in the range of 20kDa to 80 kDa.

With regard to PEGylation, it should be noted that generally, theinvention also encompasses any polypeptide of the invention that hasbeen PEGylated at one or more amino acid positions, preferably in such away that said PEGylation either (1) increases the half-life in vivo; (2)reduces immunogenicity; (3) provides one or more further beneficialproperties known per se for PEGylation; (4) does not essentially affectthe affinity of the polypeptide for CX3CR1 (e.g. does not reduce saidaffinity by more than 50%, and more preferably not by more than 10%, asdetermined by a suitable assay, such as those described in the Examplesbelow); and/or (4) does not affect any of the other desired propertiesof the polypeptides of the invention. Suitable PEG-groups and methodsfor attaching them, either specifically or non-specifically, will beclear to the skilled person.

According to a specifically preferred embodiment of the invention, aPEGylated polypeptide of the invention includes one PEG moiety of linearPEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEGmoiety is attached to the polypeptide in a linker region and,specifially, at a Cys residue, for example at position 5 of a GS8-linkerpeptide as shown in SEQ ID NO:235.

Preferred examples of PEGylated polypeptides of the invention arePEGylated preferably with one of the PEG reagents as mentioned above,such as “Sunbrighte ME-400MA” as shown in the following chemicalformula:

which has an average molecular weight of 40 kDa.

Therapeutic Uses

In one aspect, the present invention provides a polypeptide of thepresent invention or a pharmaceutical composition comprising saidpolypeptide for use as a medicament.

In one aspect, the present invention provides the use of a polypeptideof the present invention or a pharmaceutical composition comprising saidpolypeptide for the treatment or prophylaxis of cardio- andcerebrovascular atherosclerotic disorders, peripheral artery disease,restenosis, diabetic nephropathy, glomerulonephritis, human crescenticglomerulonephritis, IgA nephropathy, membranous nephropathy, lupusnephritis, vasculitis including Henoch-Schonlein purpura and Wegener'sgranulomatosis, rheumatoid arthritis, osteoarthritis, allograftrejection, systemic sclerosis, neurodegenerative disorders anddemyelinating disease, multiple sclerosis (MS), Alzheimer's disease,pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatorypain, or cancer.

In another aspect, the present invention provides the use of apolypeptide of the present invention or a pharmaceutical compositioncomprising said polypeptide for the treatment or prophylaxis ofatherosclerosis.

In another aspect, the present invention provides the use of apolypeptide of the present invention or a pharmaceutical compositioncomprising said polypeptide for the treatment or prophylaxis ofatherosclerosis by preventing and/or reducing the formation of newatherosclerotic lesions or plaques and/or by preventing or slowingprogression of existing lesions and plaques.

In another aspect, the present invention provides the use of apolypeptide of the present invention or a pharmaceutical compositioncomprising said polypeptide for the treatment or prophylaxis ofatherosclerosis by changing the composition of the plaques to reduce therisk of plaque rupture and atherothrombotic events.

In one aspect, the present invention also provides a method of treating,or reducing the risk of, cardio- and cerebrovascular atheroscleroticdisorders, peripheral artery disease, restenosis, diabetic nephropathy,glomerulonephritis, human crescentic glomerulonephritis, IgAnephropathy, membranous nephropathy, lupus nephritis, vasculitisincluding Henoch-Schonlein purpura and Wegener's granulomatosis,rheumatoid arthritis, osteoarthritis, allograft rejection, systemicsclerosis, neurodegenerative disorders and demyelinating disease,multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such asCOPD, asthma, neuropathic pain, inflammatory pain, or cancer, in aperson suffering from or at risk of, said disease or condition, whereinthe method comprises administering to the person a therapeuticallyeffective amount of a polypeptide according to the present invention ora pharmaceutical composition comprising said polypeptide.

In one aspect, the present invention also provides a method of treating,or reducing the risk of atherosclerosis in a person suffering from or atrisk of, said disease or condition, wherein the method comprisesadministering to the person a therapeutically effective amount ofpolypeptide of the present invention or a pharmaceutical compositioncomprising said polypeptide.

In one aspect, the present invention also provides a method of treating,or reducing the risk of atherosclerosis by preventing and/or reducingthe formation of new atherosclerotic lesions or plaques and/or bypreventing or slowing progression of existing lesions and plaques in aperson suffering from or at risk of, said disease or condition, whereinthe method comprises administering to the person a therapeuticallyeffective amount of polypeptide of the present invention or apharmaceutical composition comprising said polypeptide.

In one aspect, the present invention also provides a method of treating,or reducing the risk of atherosclerosis by changing the composition ofthe plaques so as to reduce the risk of plaque rupture andatherothrombotic events in a person suffering from or at risk of, saiddisease or condition, wherein the method comprises administering to theperson a therapeutically effective amount of a polypeptide of thepresent invention or a pharmaceutical composition comprising saidpolypeptide.

In one aspect, a polypeptide of the present invention is indicated foruse in the treatment or prophylaxis of a disease or disorder that isassociated with CX3CR1.

In one aspect, a polypeptide of the present invention is indicated foruse in the treatment or prophylaxis of diseases or conditions in whichmodulation of activity at the CX3CR1 receptor is desirable. In oneaspect, the present invention also provides a method of treating, orreducing the risk of, diseases or conditions in which antagonism of theCX3CR1 receptor is beneficial which comprises administering to a personsuffering from or at risk of, said disease or condition, a polypeptideof the present invention.

Prophylaxis is expected to be particularly relevant to the treatment ofpersons who have suffered a previous episode of, or are otherwiseconsidered to be at increased risk of, the disease or condition inquestion. Persons at risk of developing a particular disease orcondition generally include those having a family history of the diseaseor condition, or those who have been identified by genetic testing orscreening to be particularly susceptible to developing the disease orcondition.

In the context of the present invention, the term “prevention, treatmentand/or alleviation” not only comprises preventing and/or treating and/oralleviating the disease, but also generally comprises preventing theonset of the disease, slowing or reversing the progress of disease,preventing or slowing the onset of one or more symptoms associated withthe disease, reducing and/or alleviating one or more symptoms associatedwith the disease, reducing the severity and/or the duration of thedisease and/or of any symptoms associated therewith and/or preventing afurther increase in the severity of the disease and/or of any symptomsassociated therewith, preventing, reducing or reversing anyphysiological damage caused by the disease, and generally anypharmacological action that is beneficial to the patient being treated.

The subject to be treated will be a mammal, and more in particular ahuman being. As will be clear to the skilled person, the subject to betreated will in particular be a person suffering from, or at risk from,the diseases, disorders or conditions mentioned herein.

It will also be clear to the skilled person that the above methods oftreatment of a disease include the preparation of a medicament for thetreatment of said disease. Furthermore, it is clear that thepolypeptides of the invention can be used as an active ingredient in amedicament or pharmaceutical composition intended for the treatment ofthe above diseases. Thus, the invention also relates to the use of apolypeptide of the invention in the preparation of a pharmaceuticalcomposition for the prevention, treatment and/or alleviation of any ofthe diseases, disorders or conditions mentioned hereinabove. Theinvention further relates to a polypeptide of the invention fortherapeutic or prophylactic use and, specifically, for the prevention,treatment and/or alleviation of any of the diseases, disorders orconditions mentioned hereinabove. The invention further relates to apharmaceutical composition for the prevention, treatment and/oralleviation of the diseases, disorders or conditions mentionedhereinabove, wherein such composition comprises at least one polypeptideof the invention.

The polypeptides of the invention and/or the compositions comprising thesame can be administered to a patient in need thereof in any suitablemanner, depending on the specific pharmaceutical formulation orcomposition to be used. Thus, the polypeptides of the invention and/orthe compositions comprising the same can for example be administeredintravenously, subcutaneously, intramuscularly, intraperitoneally,transdermally, orally, sublingually (e.g. in the form of a sublingualtablet, spray or drop placed under the tongue and adsorbed through themucus membranes into the capillary network under the tongue),(intra-)nasally (e.g. in the form of a nasal spray and/or as anaerosol), topically, by means of a suppository, by inhalation,intravitreally (esp. for the treatment of dry AMD or glaucoma), or anyother suitable manner in an effective amount or dose.

The polypeptides of the invention and/or the compositions comprising thesame are administered according to a regimen of treatment that issuitable for preventing, treating and/or alleviating the disease,disorder or condition to be prevented, treated or alleviated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease, disorder or conditionto be prevented, treated or alleviated, the severity of the disease, theseverity of the symptoms thereof, the specific polypeptide of theinvention to be used, the specific route of administration andpharmaceutical formulation or composition to be used, the age, gender,weight, diet, general condition of the patient, and similar factors wellknown to the clinician. Generally, the treatment regimen will comprisethe administration of one or more polypeptides of the invention, or ofone or more compositions comprising the same, in therapeutically and/orprohylactically effective amounts or doses.

Generally, for the prevention, treatment and/or alleviation of thediseases, disorders and conditions mentioned herein and depending on thespecific disease, disorder or condition to be treated, the potency ofthe specific polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the polypeptides of the invention will generally beadministered in an amount between 0.005 and 20.0 mg per kilogram of bodyweight and dose, preferably between 0.05 and 10.0 mg/kg/dose, and morepreferably between 0.5 and 10 mg/kg/dose, either continuously (e.g. byinfusion) or as single doses (such as e.g. daily, weekly, or monthlydoses; cf. below), but can significantly vary, especially, depending onthe before-mentioned parameters.

For prophylactic applications, compositions containing the polypeptidesof the invention may also be administered in similar or slightly lowerdosages. The dosage can also be adjusted by the individual physician inthe event of any complication.

Depending on the specific polypeptide of the invention and its specificpharmacokinetic and other properties, it may be administered daily,every second, third, fourth, fifth or sixth day, weekly, monthly, andthe like. An administration regimen could include long-term, weeklytreatment. By “long-term” is meant at least two weeks and preferablymonths, or years of duration.

The efficacy of the polypeptides of the invention, and of compositionscomprising the same, can be tested using any suitable in vitro assay,cell-based assay, in vivo assay and/or animal model known per se, or anycombination thereof, depending on the specific disease involved.Suitable assays and animal models will be clear to the skilled person,and for example include the assays and animal models used in theExamples below.

For pharmaceutical use, the polypeptides of the invention may beformulated as a pharmaceutical preparation comprising (i) at least onepolypeptide of the invention and (ii) at least one pharmaceuticallyacceptable carrier, diluent, excipient, adjuvant, and/or stabilizer, and(iii) optionally one or more further pharmaceutically activepolypeptides and/or compounds. By “pharmaceutically acceptable” is meantthat the respective material does not show any biological or otherwiseundesirable effects when administered to an individual and does notinteract in a deleterious manner with any of the other components of thepharmaceutical composition (such as e.g. the pharmaceutically activeingredient) in which it is contained. Specific examples can be found instandard handbooks, such as e.g. Remington's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Company, USA (1990). For example, thepolypeptides of the invention may be formulated and administered in anymanner known per se for conventional antibodies and antibody fragmentsand other pharmaceutically active proteins. Thus, according to a furtherembodiment, the invention relates to a pharmaceutical composition orpreparation that contains at least one polypeptide of the invention andat least one pharmaceutically acceptable carrier, diluent, excipient,adjuvant and/or stabilizer, and optionally one or more furtherpharmaceutically active substances.

By means of non-limiting examples, such a formulation may be in a formsuitable for oral administration, for parenteral administration (such asby intravenous, intramuscular, subcutaneous, intrathecal,intracavernosal or intraperitoneal injection or intravenous infusion),for topical administration, for sublingual administration, foradministration by inhalation, by a skin patch, by an implant, by asuppository, for transdermal, nasal, intravitreal, rectal or vaginaladministration, and the like. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person.

Pharmaceutical preparations for parenteral administration, such asintravenous, intramuscular, subcutaneous injection or intravenousinfusion may for example be sterile solutions, suspensions, dispersions,emulsions, or powders which comprise the active ingredient and which aresuitable, optionally after a further dissolution or dilution step, forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andpharmaceutically acceptable aqueous buffers and solutions such asphysiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution; water oils; glycerol; ethanol; glycolssuch as propylene glycol, as well as mineral oils, animal oils andvegetable oils, for example peanut oil, soybean oil, as well as suitablemixtures thereof.

Solutions of the active compound or its salts may also contain apreservative to prevent the growth of microorganisms, such asantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal (thiomersal), and thelike. In many cases, it will be preferable to include isotonic agents,for example, sugars, buffers or sodium chloride. The proper fluidity canbe maintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. Other agents delaying absorption, forexample, aluminum monostearate and gelatin, may also be added.

In all cases, the ultimate dosage form must be sterile, fluid and stableunder the conditions of manufacture and storage. Sterile injectablesolutions are prepared by incorporating the active compound in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

Usually, aqueous solutions or suspensions will be preferred. Generally,suitable formulations for therapeutic proteins such as the polypeptidesof the invention are buffered protein solutions, such as solutionsincluding the protein in a suitable concentration (such as from 0.001 to400 mg/ml, preferably from 0.005 to 200 mg/ml, more preferably 0.01 to200 mg/ml, more preferably 1.0-100 mg/ml, such as 1.0 mg/ml (i.v.administration) or 100 mg/ml (s.c. administration) and an aqueous buffersuch as:

-   -   phosphate buffered saline, pH 7.4,    -   other phosphate buffers, pH 6.2 to 8.2,    -   histidine buffers, pH 5.5 to 7.0,    -   succinate buffers, pH 3.2 to 6.6, and    -   citrate buffers, pH 2.1 to 6.2,

and, optionally, salts (e.g. NaCl) and/or sugars or polyalcohols (suchas trehalose, mannitol, or glycerol) for providing isotonicity of thesolution.

Preferred buffered protein solutions are solutions including about 0.05mg/ml of the polypeptide of the invention dissolved in 25 mM phosphatebuffer, pH 6.5, adjusted to isotonicity by adding 220 mM trehalose. Inaddition, other agents such as a detergent, e.g. 0.02% Tween-20 orTween-80, may be included in such solutions. Formulations forsubcutaneous application may include significantly higher concentrationsof the polypeptide of the invention, such as up to 100 mg/ml or evenabove 100 mg/ml. However, it will be clear to the person skilled in theart that the ingredients and the amounts thereof as given above do onlyrepresent one, preferred option. Alternatives and variations thereofwill be immediately apparent to the skilled person, or can easily beconceived starting from the above disclosure.

The polypeptides of the invention may also be administered usingsuitable depot, slow-release or sustained-release formulations, e.g.suitable for injection, using controlled-release devices forimplantation under the skin, and/or using a dosing pump or other devicesknown per se for the administration of pharmaceutically activesubstances or principles. In addition, the polypeptides of the inventionmay be formulated in the form of a gel, cream, spray, drop, patch orfilm which, if placed on the skin, passes through the skin.

Also, compared to conventional antibodies or antibody fragments, onemajor advantage of the use of the polypeptides of the invention is thatthey can also be easily administered via routes other than parenteraladministration and can be easily formulated for such administration. Forexample, as described in the international application WO2004/041867,such polypeptides may be formulated for oral, intranasal, intrapulmonaryand transdermal administration.

According to another embodiment of the invention there is provided apharmaceutical combination comprising at least one polypeptide of theinvention as disclosed herein and at least one other therapeutic agentselected from the group consisting of statins, antiplatelets,anticoagulants, antidiabetics and anti hypertensives.

Such pharmaceutical combination may optionally additionally comprise adiluent, excipient, adjuvant and/or stabilizer.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition. Also, when two ormore active substances or principles are to be used as part of acombined treatment regimen, each of the substances or principles may beadministered in the same amount and according to the same regimen asused when the compound or principle is used on its own, and suchcombined use may or may not lead to a synergistic effect. However, whenthe combined use of the two or more active substances or principlesleads to a synergistic effect, it may also be possible to reduce theamount of one, more or all of the substances or principles to beadministered, while still achieving the desired therapeutic action. Thismay for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

Yet, a further embodiment of the invention is a method for treating thediseases and disorders as set out above, comprising administering to anindividual, simultaneously, separately or sequentially, an effectiveamount of at least one polypeptide of the invention and at least oneagent selected from the group consisting of a statin, an antiplatelet,an anticoagulant, an antidiabetic and an antihypertensive.

According to a further aspect of the invention, the polypeptide of theinvention is prepared to be administered in combination with other drugsused for the treatment of the diseases and disorders set out above, suchother drugs being selected from the group consisting of a statin, anantiplatelet, an anticoagulant, an antidiabetic and an antihypertensive.

According to still another aspect of the invention, drugs used for thetreatment of the diseases and disorders set out above, such drugs beingselected from the group consisting of a statin, an antiplatelet, ananticoagulant, an antidiabetic and an antihypertensive are prepared tobe administered in combination with the polypeptide of the invention.

According to a further aspect of the invention, the polypeptide of theinvention is used in combination with a device useful for theadministration of the polypeptide, such as a syringe, injector pen, orother device.

According to still another embodiment of the invention, there isprovided a method of diagnosing a disease, disorder or conditionmediated by CX3CR1 dysfunction comprising the steps of:

a) obtaining a sample from a subject, and

b) contacting, in vitro, the sample with a polypeptide of the inventionas defined above, and

c) detecting the binding of said polypeptide to said sample, and

d) comparing the binding detected in step (c) with a standard, wherein adifference in binding relative to said sample is diagnostic of adisease, disorder or condition characterised by CX3CR1 dysfunction.

According to another embodiment of the invention, there is provided amethod of diagnosing a disease, disorder or condition mediated by CX3CR1dysfunction comprising the steps of:

a) obtaining a sample from a subject, and

b) contacting the sample with a polypeptide of the invention as definedabove;

c) determining the amount of CX3CR1 in the sample; and

d) comparing the amount determined in step (c) with a standard, whereina difference in amount relative to said sample is diagnostic of adisease, disorder or condition characterised by CX3CR1 dysfunction.

The above diagnostic methods can also be used for monitoring theeffectiveness of a therapeutic treatment of a subject.

According to another embodiment of the invention, there is provided akit for diagnosing a disease, disorder or condition mediated by CX3CR1dysfunction, for use in a method as defined above, such kit comprisingat least one polypeptide of the invention and, optionally, one or moremedia, detection means and/or in vitro or in vivo imaging agents, and,further optionally, instructions of use. Suitable in vivo imaging agentsinclude 99mTc, 111Indium, 123Iodine, and, for magnetic resonanceimaging, paramagnetic compounds.

The invention further provides a kit comprising at least one polypeptideof the invention and, additionally, one or more other componentsselected from the group consisting of other drugs used for the treatmentof the diseases and disorders as described above, and devices asdescribed above.

The invention further provides methods of manufacturing a polypeptide ofthe invention, such methods generally comprising the steps of:

-   -   culturing host cells comprising a nucleic acid capable of        encoding a polypeptide of the invention (hereinafter: “nucleic        acid of the invention”) under conditions that allow expression        of the polypeptide of the invention; and,    -   recovering or isolating the polypeptide expressed by the host        cells from the culture; and    -   optionally further purifying and/or modifying and/or formulating        the polypeptide of the invention.

A nucleic acid of the invention can be genomic DNA, cDNA or syntheticDNA (such as DNA with a codon usage that has been specifically adaptedfor expression in the intended host cell or host organism). According toone embodiment of the invention, the nucleic acid of the invention is inessentially isolated form, as defined hereabove.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form. The vector mayespecially be an expression vector, i.e. a vector that can provide forexpression of the polypeptide in vitro and/or in vivo (e.g. in asuitable host cell, host organism and/or expression system). Suchexpression vector generally comprises at least one nucleic acid of theinvention that is operably linked to one or more suitable regulatoryelement(s), such as promoter(s), enhancer(s), terminator(s), and thelike. Specific examples of such regulatory elements and other elements,such as integration factor(s), selection marker(s), signal or leadersequence(s), reporter gene(s), and the like, useful or necessary forexpressing polypeptides of the invention, are disclosed e.g. on pp. 131to 133 of WO2006/040153.

The nucleic acids of the invention can be prepared or obtained in amanner known per se (e.g. by automated DNA synthesis and/or recombinantDNA technology), based on the information on the amino acid sequencesfor the polypeptides of the invention given herein, and/or can beisolated from a suitable natural source.

According to another embodiment, the invention relates to a host or hostcell that expresses or is capable of expressing a polypeptide of theinvention; and/or that contains a nucleic acid encoding a polypeptide ofthe invention. According to a particularly preferred embodiment, saidhost cells are bacterial cells, yeast cells, fungal cells or mammaliancells.

Suitable bacterial cells include cells from gram-negative bacterialstrains such as strains of Escherichia coli, Proteus, and Pseudomonas,and gram-positive bacterial strains such as strains of Bacillus,Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cellinclude cells from species of Trichoderma, Neurospora, and Aspergillus.Suitable yeast cells include cells from species of Saccharomyces (forexample Saccharomyces cerevisiae), Schizosaccharomyces (for exampleSchizosaccharomyces pombe), Pichia (for example Pichia pastoris andPichia methanolica), and Hansenula.

Suitable mammalian cells include for example CHO cells, BHK cells, HeLacells, COS cells, NSO cells, HEK cells, and the like. However, amphibiancells, insect cells, plant cells, and any other cells used in the artfor the expression of heterologous proteins can be used as well.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of immunoglobulin single variable domainpolypeptides and protein therapeutics containing them include strains ofE. coli, Pichia pastoris, and S. cerevisiae that are suitable for largescale expression, production and fermentation, and in particular forlarge scale (bio-)pharmaceutical expression, production andfermentation.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a polypeptide of theinvention for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression.

Polypeptides of the invention produced in a cell as set out above can beproduced either intracellullarly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or they can be produced extracellularly(secreted into the medium in which the host cells are cultured) and thenisolated from the culture medium and optionally further purified.

Further methods and reagents used for the recombinant production ofpolypeptides, such as suitable expression vectors, transformation ortransfection methods, selection markers, methods of induction of proteinexpression, culture conditions, and the like, are known in the art.Similarly, protein isolation and purification techniques useful in amethod of manufacture of a polypeptide of the invention are well knownto the skilled person.

Production of the polypeptides of the invention through fermentation inconvenient recombinant host organisms such as E. coli and yeast iscost-effective, as compared to conventional antibodies which alsorequire expensive mammalian cell culture facilities. Furthermore,achievable levels of expression are high and yields of the polypeptidesof the invention are in the range of 1 to 10 g/l (E. coli) and up to 10g/l (yeast) and more.

EXAMPLES

Generation CHO, Baf/3, Caki and HEK293 Cell Lines Overexpressing HumanCX3CR1 or cynomolgus CX3CR1

CHO and Baf/3 cells overexpressing human or cynomolgus CX3CR1 weregenerated using techniques known in the art. Cells expressing human CCR2or CCR5 were also generated using techniques known in the art.

The cDNA was cloned into pCDNA3.1(+)-neo for human CX3CR1 whereaspcDNA-DEST40-neo was used for mouse CX3CR1.

The amino acid sequences of humanCX3CR1 and cynomolgus CX3CR1 aredepicted in SEQ ID NO: 255 and 256, respectively.

To establish Camel Kidney (Caki) cells overexpressing human CX3CR1 ormouse CX3CR1, parental Caki cells were electroporated withpCDNA3.1(+)-neo-hCX3CR1 or pcDNA-DEST40-neo-mCX3CR1, respectively. Forall conditions, transfectants were selected by adding 1 mg/mL geneticin(Invitrogen, Carlsbad, Calif., USA).

Human Embyonic Kidney (HEK293) cells overexpressing human CX3CR1 orcynomolgus CX3CR1 were generated by lipid-mediated transfection withFugene (Roche) of pCDNA3.1(+)-neo-hCX3CR1 or cyCX3CR1 plasmids,respectively, in the HEK293 parental cell line. These cells were used astransient transfectants and as such not put under selection. In brief,2*10E6 cells were seeded per T75 and incubated overnight beforetransfection. After removal of the culture medium, cells weretransfected with the respective plasmids (9 μg) and Fugene (27 μl)according to manufacturer's instructions. 48 hours post transfection,cells were harvested and frozen for further usage.

Example 1 Immunization with CX3CR1 Induces a Humoral Immune Response inLlama

1.1. Immunizations

After approval of the Ethical Committee (University Antwerp, Belgium,UA2008A1, 2008/096, 2007/068), 9 llamas (designated No. 368, 369, 370,381, 382, 384, 312, 313 and 314) were immunized.

Six llamas (312, 313, 314, 381, 382 and 384) were immunized with 4intramuscular injections (2 mg/dose at weekly or biweekly intervals) ofpVAX1-huCX3CR1 plasmid vector (Invitrogen, Carlsbad, Calif., USA). Threellamas (381, 382 and 384) subsequently received 4 subcutaneousinjections of human CX3CR1 overexpressing Caki cells which wereestablished as described above. Cells were re-suspended in D-PBS andkept on ice prior to injection.

Three additional llamas (designated No. 368, 369 and 370) were immunizedaccording to standard protocols with 4 subcutaneous injections of humanCX3CR1 overexpressing Caki cells which were established as describedabove. Cells were re-suspended in D-PBS and kept on ice prior toinjection. Subsequently, these llamas were administered two injectionswith recombinant CX3CR1 NT/EC3 fragment coupled to BSA (Table 13).Peptides were ordered at NeoMPS (Polypeptidegroup, Strasbourg, France)and coupled to BSA according to standard protocols.

TABLE 13  Sequence of peptide fragments used for immunization boost SEQID Fragment sequence NO: CX3CR1- Ac-Met-Asp-Gln-Phe-Pro-Glu-Ser-Val- 228NT Thr-Glu-Asn-Phe-Glu-Tyr-Asp-Asp-  Leu-Ala-Glu-Ala-Cys-NH2 CX3CR1-Ac-Lys-Leu-Tyr-Asp-Phe-Phe-Pro-Ser- 229 EC3Cys-Asp-Met-Arg-Lys-Asp-Leu-Arg-Leu- NH2

The first injection was formulated in Complete Freund's Adjuvant (Difco,Detroit, Mich., USA), while the subsequent injection was formulated inIncomplete Freund's Adjuvant (Difco, Detroit, Mich., USA).

1.2. Evaluation of Induced Immune Responses in Llama

To evaluate the induction of immune responses in the animals againsthuman CX3CR1 by ELISA or FACS, sera were collected from llamas 312,313and 314 at day 0 (pre-immune), and different time points in theimmunization schedule (time of peripheral blood lymphocyte [PBL]collection).

In short, Neutravidin (24/ml) was immobilized overnight at 4° C. in a96-well Maxisorb plate (Nunc, Wiesbaden, Germany). Wells were blockedwith a casein solution (1%) in PBS. Subsequently biotinylatedrecombinant NT fragment (Polypeptide, Strasbourg, France) orbiotinylated EC3 fragments of CX3CR1 (Polypeptide, Strasbourg, France)were captured at 2 μg/ml. After addition of serum dilutions,specifically bound immunoglobulins were detected using a horseradishperoxidase (HRP)-conjugated goat anti-llama immunoglobulin (BethylLaboratories Inc., Montgomery, Tex., USA) and a subsequent enzymaticreaction in the presence of the substrate TMB One(3,3′,5,5′-tetramentylbenzidine) (Promega, Mannheim, Germany), showingthat a significant antibody-dependent immune response against CX3CR1 wasinduced after the peptide immunizations.

Additionally, serum titers of cell immunized animals were confirmed byFACS analysis on actively growing human CX3CR1 overexpressing CHO cells.The CX3CR1 serum titer responses for llamas 368, 369 and 370 weredetermined with serum sampled after 4 cell immunizations (day 49), 4cell immunizations and 1 peptide boost (day 77) and 4 cell immunizationsand 2 peptide boosts (day 81). Cells were harvested and washed beforeincubation with the serum dilutions. Detection was performed with goatanti-llama IgG (Bethyl, Montgomery, Tex., USA) followed by donkeyanti-goat coupled with PE (Jackson Laboratories, Suffolk, UK) and readout by analysis on FACSArray (BD Biosciences). A summary of the obtainedserum responses as determined by either ELISA or FACS is shown in Table14 and Table 15.

TABLE 14 Serum titer analysis for the cell/peptide immunized animalsELISA FACS Recom- Recom- After binant binant After cell peptide LlamaImmunogen NT EC3 immunization boosts 368 Caki- + +/− − − huCX3CR1 +NT/EC3 peptide 369 Caki- + +/− + + huCX3CR1 + NT/EC3 peptide 370 Caki-++ ++ − + huCX3CR1 + NT/EC3 peptide

TABLE 15 Serum titer analysis for the DNA/cell immunized animals ELISAFACS Recom- Recom- After binant binant After DNA Cell Llama Immunogen NTEC3 immunization boosts 381 DNA + Caki- ++ + ++ ++ huCX3CR1 382 DNA +Caki- + − − − huCX3CR1 384 DNA + Caki- ++ − − − huCX3CR1

For the DNA only immunized llamas (312, 313 and 314) no serum titer wasdetermined.

Example 2 Cloning of the Heavy-chain Only Antibody Fragment Repertoiresand Preparation of Phage

Following the final immunogen injection of each subset, immune tissuesas the source of B-cells that produce the heavy-chain antibodies werecollected from the immunized llamas. For llama 312,313 and 314, two150-m1 blood samples, collected 4 and 8 days after the last antigeninjection were collected per animal. For llamas 368, 369 and 370 four150 ml blood samples were collected, 5 and 7 days after the last cellimmunization and additionally 4 and 8 days after the last peptideimmunization. Next to those, two lymph node biopsies were taken, 12 daysafter the last cell immunization and 12 days after the last peptideimmunization. For llamas 381, 382 and 384 five 150 ml blood samples werecollected, 8 days after the last DNA immunization and additionally 4days after the first cell boost, 8 and 11 days after the second cellboost and 8 days after the last cell immunization. Next to those, onelymph node biopsy was taken, 8 days after the second cell immunization.

From the blood samples, peripheral blood lymphocytes (PBLs) wereprepared using Ficoll-Hypaque according to the manufacturer'sinstructions (Amersham Biosciences, Piscataway, N.J., USA). From thePBLs and the lymph node biopsy (LN), total RNA was extracted, which wasused as starting material for RT-PCR to amplify the VHH encoding DNAsegments.

For each immunized llama, libraries were constructed by pooling thetotal RNA isolated from samples originating from a certain subset of theimmunization schedule i.e. after one type of immunization antigen, andfor some llamas samples from the different animals were pooled into onelibrary (Table 16).

TABLE 16 Pooling of the different sample for library constructionLibrary Name Llama Sample 368-PBL1 + 2 + LN- 368 PBL 1 and 2, LNV-100209 369 + 370-PBL1 + 2 + 369, 370 PBL 1 and 2, LN LN-V-100209368-PBL3 + 4-V-280909 368 PBL 3 and 4 369-PBL3 + 4-V-070409 369 PBL 3and 4 370-PBL3 + 4-V-070409 370 PBL 3 and 4 381-PBL1-V-180310 381 PBL 1382-PBL1-V-180310 382 PBL1 384-PBL1-V-180310 384 PBL1 381-PBL1 + 2 + 3 +381 PBL 1, 2, 3, 4, 5 and LN 4 + 5 + LN-V-280909 382-PBL1 + 2 + 3 + 382PBL 1, 2, 3, 4, 5 and LN 4 + 5 + LN-V-280909 384-PBL1 + 2 + 3 + 384 PBL1, 2, 3, 4, 5 and LN 4 + 5 + Ln-V-280909 312 + 313 + 314- 312, 313 and314 PBL 1 and 2 PBL1 + 2-V-220210 312-PBL1 + 2-V-180310 312 PBL 1 and 2313-PBL1 + 2-V-180310 313 PBL 1 and 2 314-PBL1 + 2-V-180310 314 PBL 1and 2

In short, the PCR-amplified VHH repertoire was cloned via specificrestriction sites into a vector designed to facilitate phage display ofthe VHH library. The vector was derived from pUC119 and contains theLacZ promoter, a M13 phage gill protein coding sequence, a resistancegene for ampicillin or carbenicillin, a multiple cloning site and ahybrid gIII-pelB leader sequence (pAX050). In frame with the VHH codingsequence, the vector encodes a C-terminal c-myc tag and a His6 tag.Phage were prepared according to standard protocols and stored afterfilter sterilization at 4° C. or at −80° C. in 20% glycerol for furtheruse.

Example 3 Selection of CX3CR1 Specific VHHs Via Phage Display

VHH repertoires obtained from all llamas and cloned as phage librarywere used in different selection strategies, applying a multiplicity ofselection conditions. Variables include i) the presentation form of theCX3CR1 protein (on different cell backgrounds or on liposomes/VLPs), ii)the antigen presentation method (In solution when using cells or coatedonto plates when using VLPs), iii) the antigen concentration iv) theorthologue used (human or cynomolgus) v) the number of selection roundsand vi) different elution methods (non-specific via trypsin or specificvia the ligand Fractalkine). All solid coated phase selections were donein Maxisorp 96-well plates (Nunc, Wiesbaden, Germany).

Selections were performed as follows: CX3CR1 antigen preparations forsolid and solution phase selection formats were presented as describedabove at multiple concentrations. After 2 h incubation with the phagelibraries followed by extensive washing, bound phages were eluted withtrypsin (1 mg/mL) for 15 minutes. When trypsin was used for phageelution, the protease activity was immediately neutralized by applying0.8 mM protease inhibitor ABSF. As control, selections without antigenwere performed in parallel.

Phage outputs were used to infect E. coli which were then in turn usedto prepare phage for the next selection round (phage rescue) After thesecond round selection the phage outputs were used to infect E. coliwhich were then plated on agar plates (LB+carb+glucose^(2%)) foranalysis of individual VHH clones. In order to screen a selection outputfor specific binders, single colonies were picked from the agar platesand grown in 1 mL 96-deep-well plates. LacZ-controlled VHH expressionwas induced by adding IPTG (1 mM final) in the absence of glucose.Periplasmic extracts (in a volume of ˜80 uL) were prepared according tostandard protocols.

Example 4 Screening of Periplasmic Extracts in CX3CR1-FraktalkineCompetition FACS Assay

Periplasmic extracts were screened in a human CX3CR1/human FractalkineFACS competition assay to assess the blocking capacity of the expressedVHHs. Human CX3CR1 was presented on CHO cells overexpressing CX3CR1.Both a setup using cells harvested from an actively growing culture anda setup using frozen cells was used. As a detection reagent labeledfractalkine was used (R&D Systems, Minneapolis, Minn., USA) labeled withalexa647 (A647-Fractalkine) at a degree of labeling of 1. To setup theassay, first a titration series of the labeled fractalkine was performedon the CHO-huCX3CR1 cells in order to determine the EC50 value forbinding. Initially screening was performed at a higher concentration offractalkine (3 nM) to increase the assay robustness. To increase thesensitivity of the screening to a maximum, the EC30 concentration (1 nM)was chosen for subsequent screening. In brief 50 μl of periplasmicextract was added to 6 nM labeled fractalkine (500) and 200 000CHO-huCX3CR1 cells. After one hour incubation at 4 C, cells were washedthree times before read out was performed on a FACS Array (BectonDickinson). First a gate was set on the intact cells as determined fromthe scatter profile. Next, dead cells were gated out by theirfluorescence profile from the PI stain (Sigma, St Louis, US). Thefluorescence profile from the alexa647 label was determined for eachsample and used for calculation of blocking capacity. As controls,conditions were taken along where there was no VHH present in the periextract or a known irrelevant VHH and samples were included where excesscold fractalkine was included. For each sample the percentage block wasdetermined using the control samples to determine the assay window.

From this screening, VHHs were selected and sequence analysis revealed120 unique VHHs belonging to 3 different B-cell lineages. The totalnumber of variants found for each B-cell lineage is depicted in Table17.

TABLE 17 Selection parameters used for the identification of thehumanCX3CR1 specific VHH B-cell lineages B-cell Representative # lineageVHH ID variants libraries 9 CX3CR1BII11H11 4 368-PBL1 + 2 + LN-V-10020913 CX3CR1BII18E06 68 368-PBL1 + 2 + LN-V-100209 368-PBL3 + 4-V-280909101 CX3CR1BII66B02 48 312 + 313 + 314-PBL1 + 2-V-220210 314-PBL1 +2-V-180310

An overview of the selection procedure and performance during initialscreening is given for all VHHs in Table 18.

TABLE 18 Selection conditions and primary screening result for thehuCX3CR1 specific VHH Selections % VHH ID Family Library first roundsecond round block CX3CR1BII 9 368- BA/F3_hCX3CR1 total CHO- total 99.0PMP11H11 PBL1 + 2 + LN- (trypsin) K1_hCX3CR1 (trypsin) V-100209CX3CR1BII 13 368- BA/F3_hCX3CR1 total CHO- total 53.1 PMP18E6 PBL3 +4-V- (trypsin) K1_hCX3CR1 (trypsin) 280909 CX3CR1BII 101 312 + 313 +314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 93.8 PMP54A12 PBL1 + 2-hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314-VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 90.8 PMP54A3 PBL1 + 2- hCX3CR1hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac(2 μM) VLPs- hFrac (2 μM) 86.6 PMP54A4 PBL1 + 2- hCX3CR1 hCX3CR1V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM)VLPs- hFrac (2 μM) 92.5 PMP54A5 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U)(10U) CX3CR1BII 101 312 + 313 + 314- VLPs- total VLPs- total 68.9PMP54A7 PBL1 + 2- hCX3CR1 (trypsin) hCX3CR1 (trypsin) V-220210 (10U)(10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2μM) 92.1 PMP54B1 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U)CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM)65.3 PMP54B2 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 90.1 PMP54B3PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 +314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 92.6 PMP54B5 PBL1 + 2-hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314-VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 87.8 PMP54D5 PBL1 + 2- hCX3CR1hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314- VLPs- totalVLPs- total 64.1 PMP54D8 PBL1 + 2- hCX3CR1 (trypsin) hCX3CR1 (trypsin)V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM)VLPs- hFrac (2 μM) 96.6 PMP54F6 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U)(10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2μM) 74.7 PMP54G3 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U)CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM)74.6 PMP54H1 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 96.0 PMP54H4PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 314- VLPs-hFrac (2 μM) VLPs- hFrac (2 μM) 73.5 PMP61F10 PBL1 + 2-V- hCX3CR1hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs-hFrac (2 μM) 68.4 PMP61D1 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U)CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 94.9 PMP61D5PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs-hFrac (2 μM) VLPs- hFrac (2 μM) 70.3 PMP61E2 PBL1 + 2-V- hCX3CR1 hCX3CR1180310 (10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2μM) 96.5 PMP61F11 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U)CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 82.0 PMP61G2PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs-hFrac (2 μM) VLPs- hFrac (2 μM) 92.1 PMP61G3 PBL1 + 2-V- hCX3CR1 hCX3CR1180310 (10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2μM) 94.5 PMP61G4 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U)CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) 94.4 PMP61F4PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs-hFrac (2 μM) VLPs- hFrac (2 μM) 78.0 PMP61A11 PBL1 + 2-V- hCX3CR1hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs-hFrac (2 μM) 94.5 PMP61B2 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U)CX3CR1BII 101 314- VLPs- total VLPs- total 69.4 PMP61C9 PBL1 + 2-V-hCX3CR1 (trypsin) hCX3CR1 (trypsin) 180310 (10U) (10U) CX3CR1BII 101312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) #N/A PMP65H02PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 +314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) #N/A PMP65E11 PBL1 + 2-hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314-VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) #N/A PMP65E10 PBL1 + 2- hCX3CR1hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac(2 μM) VLPs- hFrac (2 μM) #N/A PMP65E05 PBL1 + 2- hCX3CR1 hCX3CR1V-220210 (10U) (10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM)VLPs- hFrac (2 μM) #N/A PMP65B11 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210(10U) (10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs-hFrac (2 μM) #N/A PMP65B07 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U)(10U) CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2μM) #N/A PMP65B09 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U)CX3CR1BII 101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM)#N/A PMP65H01 PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII101 312 + 313 + 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) #N/A PMP65G07PBL1 + 2- hCX3CR1 hCX3CR1 V-220210 (10U) (10U) CX3CR1BII 101 314- VLPs-hFrac (2 μM) VLPs- hFrac (2 μM) #N/A PMP66H08 PBL1 + 2-V- hCX3CR1hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs-hFrac (2 μM) #N/A PMP66H04 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U)(10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) #N/APMP66F02 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII 101314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) #N/A PMP66E11 PBL1 + 2-V-hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM)VLPs- hFrac (2 μM) #N/A PMP66D10 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310(10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM)#N/A PMP66D08 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) #N/A PMP66B02 PBL1 + 2-V-hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM)VLPs- hFrac (2 μM) #N/A PMP66A04 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310(10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM)#N/A PMP66D04 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM) #N/A PMP66D02 PBL1 + 2-V-hCX3CR1 hCX3CR1 180310 (10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM)VLPs- hFrac (2 μM) #N/A PMP66D06 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310(10U) (10U) CX3CR1BII 101 314- VLPs- hFrac (2 μM) VLPs- hFrac (2 μM)#N/A PMP66G01 PBL1 + 2-V- hCX3CR1 hCX3CR1 180310 (10U) (10U)

The amino acid sequences of all obtained unique VHHs are shown in theSequence Listing and above (CDRs and framework regions were indicated).

Example 5 Characterization of Purified VHHs

Inhibitory anti-CX3CR1 VHHs selected from the screening described inExample 4 were further purified and characterized. Selected VHHs wereexpressed in E. coli TG1 as c-myc, His6-tagged proteins. Expression wasinduced by addition of 1 mM IPTG and allowed to continue for 4 hours at3TC. After spinning the cell cultures, periplasmic extracts wereprepared by freeze-thawing the pellets. These extracts were used asstarting material and VHHs were purified via IMAC and size exclusionchromatography (SEC) resulting in 95% purity as assessed via SDS-PAGE.

Inhibition by Anti-CX3CR1 VHHs of Human Fractalkine Binding to HumanCX3CR1 Expressed on the BA/F3 Cells

The blocking capacity towards the ligand fractalkine of the VHHs wasevaluated in a human CX3CR1 competition FACS as outlined in Example 4.Either CHO-huCX3CR1 cells, BA/F3-huCX3CR1 cells or transientlytransfected HEK293T cells were used. The amount of labeled ligand usedin the different competition setups was also varied. The IC₅₀ values forVHHs blocking the interaction of human fractalkine to human CX3CR1 aredepicted in Table 19.

TABLE 19 Potency and efficacy of the VHH in a ligand competition FACSVHH ID Family Cell line IC50 % block Repeats 11H11 9 CHO-huCX3CR1 1.7E−8100 4 18E06 13 CHO-huCX3CR1 1.8E−9 33 4 54A12 101 CHO-huCX3CR1 2.1E−9104 2 54D08 101 CHO-huCX3CR1 1.5E−8 101 2 54A07 101 CHO-huCX3CR1 1.1E−878 2 54D05 101 CHO-huCX3CR1 2.7E−9 102 2 54B03 101 CHO-huCX3CR1 2.5E−8108 1 54G03 101 CHO-huCX3CR1 5.6E−8 107 1 11H11 9 BA/F3-huCX3CR1 8.1E−9100 3 18E06 13 BA/F3-huCX3CR1 2.8E−9 71 3 54A12 101 BA/F3-huCX3CR14.0E−9 100 4 54D08 101 BA/F3-huCX3CR1 3.8E−8 99 1 54A07 101BA/F3-huCX3CR1 1.5E−8 81 4 54D05 101 BA/F3-huCX3CR1 5.5E−9 99 1 54B03101 BA/F3-huCX3CR1 3.3E−8 99 1 54G03 101 BA/F3-huCX3CR1 9.8E−8 98 154A12 101 HEK293-huCX3CR1 6.8E−9 96 5 54D08 101 HEK293-huCX3CR1 8.4E−895 2 54A07 101 HEK293-huCX3CR1 2.3E−8 52 2 54D05 101 HEK293-huCX3CR15.3E−9 94 5 54B03 101 HEK293-huCX3CR1 6.7E−8 92 2 54G03 101HEK293-huCX3CR1 2.7E−7 89 2 61E02 101 HEK293-huCX3CR1 8.2E−8 98 2 61B04101 HEK293-huCX3CR1 5.7E−8 97 2 61B02 101 HEK293-huCX3CR1 1.0E−8 94 254H01 101 HEK293-huCX3CR1 1.0E−8 64 2 54A04 101 HEK293-huCX3CR1 4.9E−8100 2 61F11 101 HEK293-huCX3CR1 4.6E−8 96 2 61G03 101 HEK293-huCX3CR16.0E−8 96 2 61G04 101 HEK293-huCX3CR1 4.2E−8 96 2 66B02 101HEK293-huCX3CR1 2.5E−9 102 2 66G01 101 HEK293-huCX3CR1 1.4E−8 99 2

Inhibition by Anti-CX3CR1 VHHs of Human Fractalkine Induced Chemotaxisof BA/F3 Cells Overexpressing Human CX3CR1

To evaluate inhibition of Fractalkine induced chemotaxis, a chemotaxisassay was setup using the ChemoTx disposable chamber with 5 μm poresize(Neuroprobe, Gaithersburg, US). Cells were harvested from an activelygrowing culture and washed before use in assay medium, RPMI (Gibco,Carlsbad, US) supplemented with 0.1% BSA. The bottom chamber was filledwith 320 pM human Fractalkine in a total volume of 300 μl. Uponapplication of the membrane, 0.13E6 cells were deposited on top of themembrane in a total volume of 70 μl. Chemotaxis was allowed for 3 hoursat 37° C. in a humidified chamber with CO2. After this incubationperiod, the membrane was removed and cells in the bottom chamber wereresuspended. The amount of ATP present in the wells was determined usingthe CellTiter-Glo kit (Promega, Madison Wis., US). Read out wasperformed on an Envision (Perkin Elmer, Massachusetts, US) with thestandard settings for luminescence read out. Titration series wereperformed in triplicate and each plate contained control samples intriplicate as well. As control, a sample without VHH was included aswell as a sample where no human Fractalkine was added to the bottomchamber. A summary of the results is shown in Table 20.

TABLE 20 Potency and efficacy of the VHH in blocking the fractalkineinduced chemotaxis VHH ID Fam IC50 % block Repeats 11H11 9 2E−7 89 518E06 13 NA 17 4 54A12 101 8E−8 84 6 54D08 101 NA 33 2 54A07 101 5E−8 454 54D05 101 7E−8 81 4 54B03 101 1E−7 73 1 54G03 101 NA 40 1 66B02 1012E−8 87 2 66G01 101 4E−7 54 2

Evaluation of the Cross Reactivity of the Anti-CX3CR1 VHHs AgainstCynomolgus CX3CR1

Initially, a FACS based binding setup was used to evaluate thecynomolgus cross reactivity. For this, the VHHs were incubated with therespective cells for 30 minutes at 4° C. followed by three wash stepsand subsequently incubated with the detection reagents. As detection, amouse anti-cmyc antibody (Serotec, MCA2200) followed by a goatanti-mouse antibody coupled to PE (Jackson 115-116-071) was used, eachincubation for 30 minutes at 4° C., followed by three wash steps.Results of the assay are shown in Table 21.

TABLE 21 EC50 value for binding of the respective VHH on human CX3CR1 oron cynomolgus CX3CR1 EC50 (M) EC50 (M) VHH ID Family Human Cynomolgusratio Repeats 11H11 9 8.0E−10 NA NA 2 18E06 13 1.5E−9 1.4E−8 9 2 54A12101 3.5E−8 1.1E−7 3.3 1 54A07 101 5.4E−7 3.0E−8 0.1 1 54D05 101 8.4E−105.0E−8 59.6 1

For later identified VHHs, a human Fractalkine competition FACS was setup using human or cynomolgus CX3CR1 expressed in HEK293T cells. Both thehuman and the cynomolgus receptor was transiently transfected in HEK293Tcells and transfections were matched by the binding of the labeledligand, human fractalkine. The competition was evaluated using the EC30concentration of fractalkine and as such obtained 1050 values are a goodestimate of the Ki value, a measure for affinity (Table 22). Theexperiment was performed as described in Example 4. The ratio of the1050 values on cynomolgus monkey and human CX3CR1 was used to evaluatepotential differences in affinity for CX3CR1 in both species.

TABLE 22 Efficacy and potency of VHHs in ligand competition FACS towardshuman and cynomolgus CX3CR1 Human Cynomolgus VHH % % ID Family IC50 (M)block IC50 (M) block ratio Repeats 54A12 101 6.8E−9 96 6.7E−8 94 9.85 554D08 101 8.4E−8 95 7.2E−8 91 0.85 2 54A07 101 2.3E−8 52 2.8E−7 69 12.32 54D05 101 5.3E−9 94 6.4E−8 91 12.25 5 54B03 101 6.7E−8 92 4.7E−7 957.05 2 54G03 101 2.7E−7 89 4.7E−7 89 1.74 2 61E02 101 8.2E−8 98 4.6E−896 0.55 2 61B04 101 5.7E−8 97 8.7E−8 93 1.53 2 61B02 101 1.0E−8 944.5E−8 92 4.37 2 54H01 101 1.0E−8 64 6.8E−8 92 6.48 2 54A04 101 4.9E−8100 3.1E−8 91 0.64 2 61F11 101 4.6E−8 96 1.6E−7 95 3.58 2 61G03 1016.0E−8 96 3.0E−7 89 5.02 2 61G04 101 4.2E−8 96 2.0E−7 100 4.8 2 66B02101 2.5E−9 102 1.9E−8 97 7.5 2 66G01 101 1.4E−8 99 1.2E−7 100 8.29 2

Binding of the Anti-human CX3CR1 VHHs to Human CCR2, Human CCR5 or MouseCX3CR1

Specificity for the huCX3CR1 receptor was evaluated by performing a FACSbinding experiment on CHO-K1 parental cells or CHO cells expressinghuCCR2, huCCR5 or msCX3CR1. The VHHs were incubated with the respectivecell lines for 30 minutes at 4° C. followed by three wash steps andsubsequently incubated with the detection reagents. As detection, amouse anti-cmyc antibody (Serotec, MCA2200) followed by a goatanti-mouse antibody coupled to PE (Jackson 115-116-071) was used, eachincubation for 30 minutes at 4° C., followed by three wash steps. Foreach cell line a quality control with receptor-specific antibody wasincluded. In addition, the highest concentration of each VHH was alsoincubated with CHO cells expressing huCX3CR1 as a positive control. Nobinding to msCX3CR1, huCCR2 or huCCR5 could be observed.

Determination of the Epitope Bin

A competitive binding experiment was setup in order to determine whetherthe VHHs bind overlapping epitopes on CX3CR1. For this, the VHH 66602labeled with alexa647 was used in a competition FACS on the BA/F3 cellsexpressing huCX3CR1. Representative VHHs from the three functionalfamilies were used as competitors for the binding of the labeled 661302.The obtained 1050 values are shown in Table 23.

TABLE 23 Competition FACS based epitope binning VHH ID family IC50 (M) %block 11H11 9 4.9E−09 100 18E06 13 2.3E−09 100 66B02 101 1.5E−09 100

As a complete inhibition of 661302 binding could be obtained by allrepresentative VHHs from the different ligand blocking families, it canbe concluded that all functional families bind in close enough proximityof each other such that they compete with binding of 661302.

Example 6 Formatting of VHHs to Bivalency

Construction of Bivalents

In order to increase potency and/or efficacy from a selection of theobtained VHHs, bivalent molecules were constructed by geneticengineering. Two VHHs were genetically linked together with a 35GSlinker in between the two building blocks and subsequently expressed inE. coli as described above for the monovalent VHHs. Different bivalentconstructs were made as listed in Table 24.

TABLE 24 Representative bivalent formats Construct ID VHH identityFamily Linker VHH identity Family CX3CR1BII007 CX3CR1BII11H11 9 35GSCX3CR1BII18E6 13 CX3CR1BII009 CX3CR1BII18E6 13 35GS CX3CR1BII11H11 9CX3CR1BII012 CX3CR1BII54D08 101 35GS CX3CR1BII18E06 101 CX3CR1BII016CX3CR1BII54A12 101 35GS CX3CR1BII54A12 101 CX3CR1BII017 CX3CR1BII54D5101 35GS CX3CR1BII54D5 101 CX3CR1BII018 CX3CR1BII66B02 101 35GSCX3CR1BII66B02 101 CX3CR1BII019 CX3CR1BII66G01 101 35GS CX3CR1BII66G01101 CX3CR1BII020 CX3CR1BII54B5 101 35GS CX3CR1BII54B5 101 CX3CR1BII026CX3CR1BII11H11 9 35GS CX3CR1BII66B02 101 CX3CR1BII027 CX3CR1BII11H11 935GS CX3CR1BII54B5 101

Inhibition by Anti-CX3CR1 VHHs of Human Fractalkine Binding to HumanCX3CR1 Expressed on the BA/F3 Cells

The inhibition of ligand binding to human CX3CR1 was investigated forthe different formats as described in Example 4. For thischaracterization the BA/F3-huCX3CR1 cell line was used showing stableexpression of the human CX3CR1 receptor. The alexa647 labeled ligandfractalkine was used at its EC30 concentration and thereby obtained 1050values are reflective of the Ki values. An overview of the obtained datais shown in Table 25.

TABLE 25 Potency of bivalent formats in ligand competition Construct IDCell line IC50 (M) % block Repeats CX3CR1BII007 CHO-huCX3CR1 3.8E−10 1002 CX3CR1BII009 CHO-huCX3CR1 7.0E−10 91 2 CX3CR1BII012 CHO-huCX3CR18.0E−10 93 1 CX3CR1BII016 HEK293-huCX3CR1 1.9E−10 102 2 CX3CR1BII017HEK293-huCX3CR1 3.1E−10 99 2 CX3CR1BII018 HEK293-huCX3CR1 3.0E−10 102 2CX3CR1BII019 HEK293-huCX3CR1 2.9E−10 100 2 CX3CR1BII020 HEK293-huCX3CR12.2E−10 102 2 CX3CR1BII026 BA/F3-huCX3CR1 7.0E−10 100 3 CX3CR1BII027BA/F3-huCX3CR1 6.7E−10 100 3

Inhibition by Anti-CX3CR1 VHHs of Human Fractalkine Induced Chemotaxisof BA/F3 Cells Overexpressing Human CX3CR1

Similar to what was described for the monovalent anti-CX3CR1 VHHs, theinhibition of fractalkine induced chemotaxis on the BA/F3-huCX3CR1 cellswas evaluated for the bivalent constructs. An identical assay setup wasused as described above and the obtained results are summarized in Table26.

TABLE 26 Inhibition of fractalkine induced chemotaxis by bivalent VHHconstructs Construct ID Cell line IC50 (M) % block Repeats CX3CR1BII007BA/F3-huCX3CR1 4E−9 101 5 CX3CR1BII009 BA/F3-huCX3CR1 2E−8 79 5CX3CR1BII012 BA/F3-huCX3CR1 4E−9 78 1 CX3CR1BII016 BA/F3-huCX3CR1 2E−988 3 CX3CR1BII017 BA/F3-huCX3CR1 3E−9 89 3 CX3CR1BII018 BA/F3-huCX3CR1 6E−10 98 6 CX3CR1BII019 BA/F3-huCX3CR1 2E−9 85 3 CX3CR1BII020BA/F3-huCX3CR1 2E−9 85 3 CX3CR1BII026 BA/F3-huCX3CR1  3E−10 98 1CX3CR1BII027 BA/F3-huCX3CR1  9E−10 98 1

Evaluation of the Cross Reactivity of the Anti-CX3CR1 VHHs AgainstCynomolgus CX3CR1

Also for the bivalent constructs the cross reactivity towards cynomolgusCX3CR1 was evaluated and compared with the human reactivity. Asdescribed earlier, either a binding setup (Table 27) or a ligandcompetition setup (Table 28) were applied using transient transfectedHEK293T cells. Batches of transient transfected cells were matched bytheir receptor expression level.

TABLE 27 Binding of bivalent constructs to human or cynomolgus CX3CR1EC50 (M) EC50 (M) Construct ID Cell line Human Cynomolgus ratio RepeatsCX3CR1BII007 HEK293T 3.1E−10 4.8E−8 154 2 CX3CR1BII009 HEK293T 2.0E−96.8E−9 3.3 2 CX3CR1BII012 HEK293T 5.6E−11 6.3E−11 1.1 1

TABLE 28 Ligand competition of bivalent constructs on human orcynomolgus CX3CR1 Human Cynomolgus % % Construct ID Cell line IC50 (M)block IC50 (M) block ratio Repeats CX3CR1BII016 HEK293T 1.9E−10 1026.9E−10 98 3.67 2 CX3CR1BII017 HEK293T 3.1E−10 99 1.6E−9  95 5.34 2CX3CR1BII018 HEK293T 3.0E−11 102 1.0E−10 97 3.33 2 CX3CR1BII019 HEK293T2.9E−10 100 8.2E−10 96 2.86 2 CX3CR1BII020 HEK293T 2.2E−10 102 3.2E−1097 1.47 2

Example 7 Exploration of Linker Length and Half Life Extension

Evaluation of the Linker Length and Positioning of the alb11 VHH

As the linker length used in a bivalent format can impact drastically onthe obtained potency, different linker lengths were evaluated.

In addition, Alb11, a Nanobody binding to human serum albumin wasincluded to increase the in vivo half-life of the formatted molecules(WO 06/122787). Different formats were made including variations on thelinker lengths used, but also the positioning of the different composingVHHs. A summary of the explored formats is shown in Table 29.

TABLE 29 Exploration of half life extension and linker length ConstructID VHH identity Linker VHH identity Linker VHH identity CX3CR1BII032CX3CR1BII66B02 9GS CX3CR1BII66B02 9GS Alb11 CX3CR1BII034 CX3CR1BII66B0235GS CX3CR1BII66B02 9GS Alb11 CX3CR1BII036 CX3CR1BII66B02 9GS Alb11 9GSCX3CR1BII66B02 CX3CR1BII040 CX3CR1BII66B02 9GS CX3CR1BII66B02 35GS Alb11CX3CR1BII041 CX3CR1BII66B02 35GS CX3CR1BII66B02 35GS Alb11 CX3CR1BII042CX3CR1BII66B02 35GS Alb11 35GS CX3CR1BII66B02

Coding sequences for the formatted VHH were cloned into an in-houseconstructed plasmid allowing expression in Pichia pastoris and secretioninto the cultivation medium. The expression vector was derived frompPICZa (Invitrogen) and contained the AOX1 promotor for tightlyregulated, methanol induced expression, a restance gene for Zeocin™, amulticloning site and the α-factor secretion signal. Upon transformationexpression cultures were grown and VHH expression was induced byaddition of methanol and allowed to continue for 48 hours at 30° C.

The potency of these different formats was evaluated using the ligandcompetition assay as described above. Seeing that the ligandconcentration used is below the EC50 value, the obtained IC50 values areequivalent to the Ki values. The obtained Ki for the different formatsis summarized in Table 30.

TABLE 30 Potency of half life extended formats in ligand competitionConstruct ID Cell line IC50 (M) % block Repeats CX3CR1BII032BA/F3-huCX3CR1 5.8E−10 99 2 CX3CR1BII034 BA/F3-huCX3CR1 5.2E−10 99 2CX3CR1BII036 BA/F3-huCX3CR1 5.6E−10 99 2 CX3CR1BII040 BA/F3-huCX3CR15.9E−10 104 1 CX3CR1BII041 BA/F3-huCX3CR1 6.4E−10 102 1 CX3CR1BII042BA/F3-huCX3CR1 8.9E−10 100 1

Impact of Human Serum Albumin on the Potency

The binding of human serum albumin (HSA) to the alb11 VHH could impacton the potency of the format and therefore ligand competition wasrepeated in presence of HSA. Briefly, to allow the binding of HSA to thealb11 VHH, the constructs under evaluation and fractalkine werepre-incubated with HSA for 30 minutes before addition to the cells. Alsothe cells were resuspended in FACS buffer supplemented with HSA. Thefinal concentration HSA used was a 50 fold excess above the highest VHHconcentration used. Subsequently, competition was allowed for 2 hoursand further processing was as described in Example 4.

Construct ID Cell line IC50 (M) % block Repeats CX3CR1BII032BA/F3-huCX3CR1 1.4E−9 100 2 CX3CR1BII034 BA/F3-huCX3CR1 1.3E−9 100 2CX3CR1BII036 BA/F3-huCX3CR1 1.4E−9 100 2

The potential interference of HSA was also evaluated in an adaptedchemotaxis setup, including HSA in the different compartments of theassay. The concentration HSA used was again a 50 fold excess over thehighest concentration of construct used and constructs were loaded withHSA for 30 minutes before start of the assay. The assay buffer was alsosupplemented with HSA such that HSA is present during the entire span ofthe experiment. As described above, the disposable ChemoTx chamber with5 μm poresize (Neuroprobe, Gaithersburg, Md., USA) was used. Cells wereharvested from an actively growing culture and washed before use inassay medium, RPMI (Gibco, Carlsbad, US) supplemented with 0.1% BSA and62.5 μM HSA (Sigma, A8763). The bottom chamber was filled with 320 pMhuman Fractalkine in a total volume of 300 μl. Upon application of themembrane, 0.13E6 cells were deposited on top of the membrane in a totalvolume of 70 μl. Chemotaxis was allowed for 3 hours at 37 C in ahumidified chamber with CO2. After this incubation period, the membranewas removed and cells in the bottom chamber were resuspended.

The amount of ATP present in well was determined using the CellTiter-Glokit (Promega, Madison Wis., USA). Read out was performed on an Envision(Perkin Elmer, Waltham, Mass., USA) with the standard factory settingsfor luminescence read out. Titration series were performed in triplicateand each plate contained control samples in triplicate as well. Ascontrol, a sample without VHH was included as well as a sample where nohuman Fractalkine was added to the bottom chamber. The obtained 1050values are listed in Table 31.

TABLE 31 Fractalkine induced chemotaxis in the presence of HSA ConstructID Cell line IC50 (M) % block Repeats CX3CR1BII032 BA/F3-huCX3CR1 6E−1098 2 CX3CR1BII034 BA/F3-huCX3CR1 9E−10 100 2 CX3CR1BII036 BA/F3-huCX3CR16E−10 102 2

Inhibition of Fractalkine Internalization by the Formatted BivalentHalf-life Extended Polypeptides

Additional functional assays were performed to demonstrate theantagonist activity of the bivalent half-life extended polypeptides. Thepolypeptides were evaluated for their ability to inhibit theinternalization of A647-Fractalkine in CHO huCX3CR1 cells. Briefly, 1E4cells/well were plated in black clear bottom, 96 well plates (BD,Franklin Lakes, N.J., USA) and grown overnight. The cells were washedonce and then equilibrated in assay buffer (HBSS with calcium andmagnesium (Gibco) supplemented with 10 mM HEPES and 0.1% BSA). Theformatted polypeptide constructs were added and the plates wereincubated for 15 minutes at 37 C. A647-Fractalkine was then added at afinal concentration of 8 nM and the cells were incubated for 60 minutesat 37 C. The media was removed and the cells were fixed for 10 minuteswith 3.7% formaldehyde solution (Polysciences, Warrington, Pa., USA).The cells were rinsed once with PBS and the nuclei were labeled withHoechst dye (Life Technologies, Grand Island, N.Y., USA). To quantitatethe internalized labeled Fractalkine, the cells were imaged using the BDPathway bioimaging system. Image segmentation was performed byidentifying the labeled cell nucleus and drawing a 3 pixel ring aroundthat mask. Mean A647 intensity was measured in the cytoplasmic ring. Theformatted polypeptides potently inhibited Fractalkine internalization assummarized in Table 32:

TABLE 32 Inhibition of A647-Fractalkine Internalization Construct IDCell line IC50 (M) Repeats CX3CR1BII032 CHO-huCX3CR1 4.0E−10 5CX3CR1BII034 CHO-huCX3CR1 7.4E−10 3 CX3CR1BII036 CHO-huCX3CR1 4.9E−10 8CX3CR1BII040 CHO-huCX3CR1 1.0E−9  3 CX3CR1BII041 CHO-huCX3CR1 1.1E−9  2CX3CR1BII042 CHO-huCX3CR1 8.5E−10 2

An Anti-CX3CR1 Formatted Bivalent Half-life Extended Polypeptide isDevoid of Agonist Activity

In order to confirm that a bivalent anti-CX3CR1 half-life extendedpolypeptide did not have agonist activity, CX3CR1B036 was evaluated forinduction of calcium influx in the CHO huCX3CR1 cells. Fractalkinemediated increases in cytosolic calcium levels in these cells in aCX3CR1 dependent manner and CX3CR1BII036 inhibited this response.

The CHO huCX3CR1 cells were plated at 5E4 cells/well in black clearbottom, 96 well plates (BD) and grown overnight. The cells wereincubated with Calcium-4 dye/2 mM probenicid (Molecular Devices,Sunnyvale, Calif., USA) in HBSS supplemented with 20 mM HEPES for 60minutes at 37° C. For demonstrating polypeptide antagonism, CX3CR1BII036was preincubated with the cells for 15 minutes prior to the addition ofFractalkine at its EC80 value. Calcium mobilization was monitored on aFLIPR Tetra system (Molecular Devices) as per the manufacturer'sinstructions. For determining agonism, there was no preincubation withthe polypeptide and instead, CX3CR1611036 was used in place ofFractalkine stimulation. While CX3CR1BII036 inhibited Fractalkinemediated calcium influx with an 1050 of 1.3 nM, no increase in cytosoliccalcium levels were observed when the polypeptide alone was added atconcentrations up to 1 μM.

Example 8 Exploration of Half Life Extension Formats Using Mouse Fc

To investigate alternative half-life extension modalities, the 66B02 VHHdomain was produced as a fusion protein with a mouse IgG2 Fc domain(66B02-mFc). An aspartic acid to alanine mutation (D265A) wasincorporated in the CH2 domain to abrogate potential Fc-mediatedeffector function in this construct (Baudino, J. Immunol., 181,6664-6669 (2008)). 66B02-mFc was expressed in HEK293T cells or NSO cellsand purified by Protein A affinity chromatography followed by ionexchange chromatography. This molecule was tested for activity utilizingthe assay formats described in Example 7. The results are summarized inTable 33:

TABLE 33 66B02-mFc Activity Assay Cell line IC50 (M) Repeats LigandBA/F3-huCX3CR1 5.7E−10 2 competition Chemotaxis BA/F3-huCX3CR1 8.9E−10 3Ligand CHO-huCX3CR1 5.2E−10 5 internalization Calcium influxCHO-huCX3CR1 9.8E−10 5

While 66B02-mFc potently inhibited Fractalkine mediated CX3CR1activation, it did not display agonist activity. No increase incytosolic calcium levels was observed with treatment with up to 1 μM ofthis molecule.

Example 9 Inhibition of Plaque Progression in a Mouse AtherosclerosisModel Bivalent Half-life Extended Polypeptides

Generation of Human CX3CR1 Knock-in Apo E^(−/−) Mice

Given the lack of cross reactivity of the identified VHHs for mouseCX3CR1 (Example 5), a human CX3CR1 knock-in mouse line (hu CX3CR1 KI)was generated at TaconicArtemis (Koeln, Germany) to enable testing ofthese molecules in mouse disease models. A strategy was employed thatallowed the expression of the human chemokine receptor under the controlof the corresponding mouse promoter while disrupting the expression ofthe endogenous mouse protein. Briefly, a targeting vector wasconstructed where the mouse CX3CR1 coding region in exon 2 was replacedwith the complete human CX3CR1 open reading frame and flanked byselection markers and IoxP sites. The targeting vector was introducedinto mouse ES cells and clones that had successfully undergonehomologous recombination were used to generate chimeric mice. These micewere bred to highly efficient Flp-deleter mice to achieve removal of theselection marker and germline transmission. The resulting hu CX3CR1 KImice in a C57BL/6 background were then crossed to Apo E^(−/−) mice (TheJackson Laboratory, Bar Harbor, Me., USA) to generate hu CX3CR1 KI ApoE^(−/−) mice. The Apo E^(−/−) mouse model provides a robust method toelicit extensive atherosclerotic plaque formation that is grosslysimilar to the human disease with respect to the site-specificlocalization of plaque formation, histological composition, and theknown risk factors (cholesterol, inflammation, hypertension, etc).

Evalulation of the Anti-CX3CR1 Bivalent Half-life Extended Polypeptidesin the Mouse Apo E^(−/−) Atherosclerosis Model

Female hu CX3CR1 KI ApoE^(−/−) mice were fed a high fat/high cholesteroldiet containing 1.5% cholesterol for 16 weeks beginning at four weeks ofage. After 10 weeks, the animals were administered by i.p. injectionvehicle (20 mM NaCitrate pH 6.0, 115 mM NaCl), 10 mg/kg 66B02-mFc onceor twice per week or 30 mg/kg CX3CR1BII036 twice per week for 6 weeks.The animals were anesthetized by gas anaesthesia and perfused with 0.9%saline. The descending aorta to the ileac bifurcation was carefullyremoved and fixed in formalin. It was then opened longitudinally, andstained with Sudan IV for 15 minutes, followed by 70% methanol for 2minutes. The vessels were washed under running water and covered withPBS. The tissues were photographed with a digital camera using SPOTAdvanced software (SPOT Imaging Solutions, Sterling Heights, Mich.,USA). The percentage of lipid staining was determined with imageanalysis software (Image-Pro Plus, MediaCybernetics, Rockville, Md.,USA) and expressed as a percentage positive staining of the vessel. Theresults from this study are summarized in Table 34:

TABLE 34 Quantification of Plaque Size in the Descending Aorta in femalehu CX3CR1 KI Apo E^(−/−) Mice % Reduction % Plaque in Plaque Group Dose# Animals Area Area Control (10 N/A 6 3.4 N/A weeks) Control (16 Vehicle17 14.8 N/A weeks) 66B02-mFc 10 mg/kg 17 13.0 16 (1× week) 66B02-mFc 10mg/kg 17 10.3 39 (p < 0.05) (2×/week) CX3CR1BII036 30 mg/kg 17 10.1 41(p < 0.05) (2×/week)

Both 66B02-mFc and CX3CR1BII036 significantly inhibited plaqueprogression when dosed twice weekly. This correlated with coverage asplasma levels of these molecules could be confirmed to be maintainedthroughout the study. For once weekly dosing of 66B02-mFc, detectableplasma levels were not maintained and this correlated with the lack ofsignificant efficacy observed after 6 weeks of treatment. Neithermolecule significantly affected plasma cholesterol or triglyceridelevels.

Example 10 Sequence Optimization of the Parental VHH

In general, during VHH sequence optimization, parental wild type VHHsequences are mutated to yield VHH sequences that are more identical tohuman VH3-JH germline consensus sequences. Specific amino acids in theframework regions that differ between the VHH and the human VH3-JHgermline consensus are altered to the human counterpart in such a waythat the protein structure, activity and stability are kept intact. Toinvestigate this, all sequence optimization variants were compared withthe parental VHH in three different assays: (i) determination of themelting temperature (Tm) in a Thermal Shift Assay (TSA), (ii) analysisof in vitro potency in fractalkine competition FACS, and for someconstructs (iii) analysis of in vitro potency in the fractalkine inducedchemotaxis assay.

Mutation of Framework Residues

For sequence optimization, the following mutations were investigated:E1D, S11L, A14P, E16G, R44Q, D46E, A74S, K83R and Q108L. The individualmutants that were generated in the parental sequence of CX3CR1BII666B02are depicted in Table 35:

TABLE 35 Investigated mutations during sequence optimization of 66B02Clone number Mutations introduced C100CX3CR1BII043 A14P, A74S, K83R,Q108L C100CX3CR1BII045 E1D, A14P, A74S, K83R, Q108L C100CX3CR1BII047S11L, A14P, A74S, K83R, Q108L C100CX3CR1BII048 A14P, E16G, A74S, K83R,Q108L C100CX3CR1BII049 A14P, R44Q, A74S, K83R, Q108L C100CX3CR1BII050A14P, D46E, A74S, K83R, Q108L C100CX3CR1BII061 S11L, A14P, E16G, A74S,K83R, Q108L C100CX3CR1BII056 S11L, A14P, E16G, R44Q, A74S, K83R, Q108LC100CX3CR1BII057 S11L, A14P, E16G, D46E, A74S, K83R, Q108LC100CX3CR1BII060 S11L, A14P, E16G, R44Q, D46E, A74S, K83R, Q108L

All constructs were cloned in an E. coli expression vector, andexpressed in E. coli as myc/His-tagged proteins in a culture volume of0.25 L to 0.5 L TB medium. Expression was induced by addition of 1 mMIPTG and allowed to continue for 4 hours at 37° C. and 250 rpm. Cellswere pelleted, and periplasmic extracts were prepared by freeze-thawingand resuspension in dPBS. These extracts were used as starting materialfor immobilized metal affinity chromatography (IMAC) using Histrap FFcrude columns (GE healthcare). Nanobodies were eluted from the columnwith 250 mM imidazole and subsequently desalted towards dPBS. The purityand integrity of Nanobodies was verified by reducing SDS-PAGE.

As summarized in Table 36, A14P, A74S, K83R and Q108L mutations had noclear effect on potency as determined from competition FACS. Similarly,the additional mutations E1D, S11L and E16G did not affect potency. Theintroduction of either R44Q or D46E on the other hand resulted in asignificant drop in potency that was even more pronounced if bothmutations were introduced.

TABLE 36 Potency of sequence optimization constructs determined byligand competition FACS Clone number IC50 % block Tm at pH 7CX3CR1BII66B02 2.6E−09 101.0 65.66 C100CX3CR1BII043 2.2E−09 101 66.49C100CX3CR1BII045 2.2E−09 101.2 66.07 C100CX3CR1BII047 2.3E−09 101.266.49 C100CX3CR1BII048 1.9E−09 101.2 67.74 C100CX3CR1BII049 1.8E−08101.2 66.07 C100CX3CR1BII050 1.7E−08 101.1 71.90 C100CX3CR1BII0611.4E−09 98.9 68.57 C100CX3CR1BII056 1.6E−08 101.1 68.57 C100CX3CR1BII0571.4E−08 99.4 74.39 C100CX3CR1BII060 1.9E−07 98.4 74.81

Also the melting temperature, predictive for the stability of the VHH,was evaluated. Most individual mutations had limited to no effect exceptfor the D46E mutation which raised the melting temperature byapproximately 6° C. The introduction of the combined mutations alsoenhanced the thermal stability, cfr 057 and 060.

Due to the major effects on the potency in ligand competition FACS, themutations R44Q and D46E were not included in the final sequence.

Mutation of CDR Residues

Based on the in silico analysis of the parental sequence, aglycosylation site was predicted at position 52. Therefore two librarieswere constructed; one for position 52 and one for position 53, which wasdesigned to include all possible amino acids at the respective position.The libraries were screened as periplasmic extracts in a ligandcompetition FACS. First, a dilution series was made of periplasmicmaterial from the parental sequence and three dilutions were selectedfor further screening. A first dilution point (two fold) was chosen togive full block of the ligand interaction whereas the other two dilutionpoints (128 and 512 fold) should result in 70% and 40% blockrespectively. Upon production of the periplasmic extracts from thelibrary, all samples were split in two and one of them was subjected toa heat treatment. Both the non-treated and the heat treated samples weresubsequently analyzed in the ligand competition FACS at the threedilution points. The impact of the mutation could be estimated bycomparing the obtained blockage with that from the parental sequence.The analysis of the heat treated samples provides a measure for apotential impact on stability of the mutation.

Based upon the initial screening results, seven mutations were selectedfor further characterization. The obtained potency in ligand competitionFACS is shown in Table 37.

TABLE 37 Removal glycosylation site at position 52 Construct IC50 (M) %block Tm at pH 7 C100CX3CR1BII66B02 2.5E−09 98.0 65.66 CX3CR1BII66B02(N52S, Q108L) 1.7E−09 98.0 66.07 CX3CR1BII66B02 (N52Q, Q108L) 2.1E−0997.9 59.83 CX3CR1BII66B02 (N52G, Q108L) 1.1E−09 98.0 59.83CX3CR1BII66B02 (N52T, Q108L) 2.8E−09 98.0 66.07 CX3CR1BII66B02 (S53T,Q108L) 1.3E−09 98.1 66.07 CX3CR1BII66B02 (S53G, Q108L) 1.2E−09 98.364.83 CX3CR1BII66B02 (S53P, Q108L) 8.0E−10 98.2 66.91

From this analysis, sequence alignment with the human reference sequenceand based upon an in silico T cell epitope recognition predictionprogram, it was decided to include the mutations N52S and S53T in thesequence.

Because of stability reasons an additional library was made for position32. The ligand competition screening was set up in a similar fashion asdescribed above. Again three dilutions of the periplasmic extracts werescreened and the obtained % block was compared with that obtained forthe parental sequence. Upon analysis of the various mutants, thesubstitution of N32T was chosen and included in the final sequenceoptimized variant.

Example 11 Analysis of the Optimized Variants

In a final characterization round the constructs listed in Table 38 werecharacterized.

TABLE 38 Sequence optimized variants of the lead VHH 66B02 Clone numberMutation introduced SEQ ID NO: CX3CR1BII00306 CX3CR1BII66B02(E1D, S11L,A14P, E16G, N32T, N52S, 138 A74S, K86R, Q113L) CX3CR1BII00307CX3CR1BII66B02(E1D, S11L, A14P, E16G, N32T, N52S, 139 S53T, A74S, K86R,Q113L) CX3CR1BII00308 CX3CR1BII66B02(E1D, S11L, A14P, E16G, A74S, 140K86R, Q113L) CX3CR1BII00312 CX3CR1BII66B02(E1D, S11L, A14P, E16G, N32T,N52S, 225 A74S, K86R, Q113L)-9GS-Alb11-9GS- CX3CR1BII66B02(S11L, A14P,E16G, N32T, N52S, A74S, K86R, Q113L) CX3CR1BII00313 CX3CR1BII66B02(E1D,S11L, A14P, E16G, N32T, N52S, 226 S53T, A74S, K86R,Q113L)-9GS-Alb11-9GS- CX3CR1BII66B02(S11L, A14P, E16G, N32T, N52S, S53T,A74S, K86R, Q113L) CX3CR1BII00314 CX3CR1BII66B02(E1D, S11L, A14P, E16G,A74S, 227 K86R, Q113L)-9GS-Alb11-9GS- CX3CR1BII66B02(S11L, A14P, E16G,A74S, K86R, Q113L)

A competition FACS experiment was performed as described above as wellas a determination of the melting temperature. The obtained values arerepresented in Table 39.

TABLE 39 Competition FACS and Tm of the sequence optimized variantsConstruct IC50 (M) % block Tm at pH 7 C100CX3CR1BII66B02 2.5E−09 98.065.05 CX3CR1BII00306 1.7E−09 97.0 68.54 CX3CR1BII00307 1.9E−09 97.068.13 CX3CR1BII00308 1.6E−09 97.0 68.13 CX3CR1BII00312 4.6E−10 100.059.37 CX3CR1BII00313 4.0E−10 100.0 58.88 CX3CR1BII00314 6.5E−10 100.058.40

These constructs were also characterized in fractalkine inducedchemotaxis as described above (Table 40).

TABLE 40 Ligand induced chemotaxis with sequence optimized variantsConstruct IC50 (M) % block n C100CX3CR1BII66B02 3.6E−08 91 3CX3CR1BII00306 6.3E−08 95 2 CX3CR1BII00307 6.3E−08 100 2 CX3CR1BII003084.4E−08 89 2 CX3CR1BII00312 2.7E−09 99 3 CX3CR1BII00313 2.7E−09 99 3CX3CR1BII00314 3.6E−09 100 3

Selected constructs were evaluated for inhibition of A647-Fractalkineinduced internalization in CHO huCX3CR1 cells. The results aresummarized in Table 41:

TABLE 41 Ligand induced internalization with sequence optimized variantsConstruct IC50 (M) n CX3CR1BII00312 5.5E−10 1 CX3CR1BII00313 3.3E−10 6

A Sequence Optimized Anti-CX3CR1 Half-life Extended Polypeptide isDevoid of Agonist Activity

In order to confirm that sequence optimized anti-CX3CR1 half-lifeextended polypeptide does not have agonist activity, CX3CR1BII00313 wasevaluated for induction of calcium influx in the CHO huCX3CR1 cells.While preincubation with CX3CR1BII00313 inhibited Fractalkine-mediatedcalcium influx with an 1050 of 1.3 nM, no increase in cytosolic calciumlevels were observed when the polypeptide alone was added atconcentrations up to 1 μM.

Example 12 Exploration of Half Life Extension Formats Using Human Fc

To investigate additional half-life extension modalities, theCX3CR1BII00306 and CX3CR1BII00307 sequence optimized VHH domains wereproduced as fusion proteins with a human IgG1 Fc domain (306D-hFc and307D-hFc). Two mutations were incorporated in the CH2 domain to abrogatepotential Fc-mediated effector function in this construct. 306D-hFc and307D-hFc were expressed in HEK293T cells or NSO cells and purified byProtein A affinity chromatography followed by ion exchangechromatography. These molecules were tested for functional activityutilizing the assay formats described in Example 7. The results aresummarized in Table 42:

TABLE 42 Activity of hFc Fusion Proteins 307D- 307D- 306D-hFc 306D-hFchFc hFc Assay Cell line IC50 Repeats IC50 Repeats Ligand BA/F3-huCX3CR16.9E−10 2 7.0E−10 2 competition Chemotaxis BA/F3-huCX3CR1 2.9E−9  23.0E−9  3 Ligand CHO-huCX3CR1 4.8E−10 3 3.7E−10 3 internalizationCalcium influx CHO-huCX3CR1 1.3E−9  3 3.2E−9  3

While these molecules potently inhibited Fractalkine mediated CX3CR1activation, they did not display agonist activity. No increases incytosolic calcium levels were observed with treatment with up to 1 μM ofthese Nanobodies alone.

Example 13 Inhibition of Plaque Progression in a Mouse AtherosclerosisModel by a Sequence Optimized Anti-CX3CR1 Nanobody

Female hu CX3CR1 KI ApoE^(−/−) mice were fed a high fat/high cholesteroldiet containing 1.5% cholesterol for 16 weeks beginning at four weeks ofage. After 10 weeks, the animals were administered by i.p. injectionvehicle (20 mM NaCitrate pH 6.0, 115 mM NaCl), 30 mg/kg CX3CR1 BII00313once or twice per week or 30 mg/kg CX3CR1BII036 twice per week for 6weeks. The animals were sacrificed and the percentage of plaque area inthe descending aorta was quantitated as described above. The resultsfrom this study are summarized in Table 43:

TABLE 43 Quantification of Plaque Size in the Descending Aorta in femalehu CX3CR1 KI Apo E^(−/−) Mice % Reduction % Plaque in Plaque Group Dose# Animals Area Area Control (10 N/A 6 2.1 N/A weeks) Control (16 Vehicle18 12.0 N/A weeks) CX3CR1BII00313 30 mg/kg) 17 10.7 13 (1× week)CX3CR1BII00313 30 mg/kg 18 5.9 62 (p < 0.01) (2×/week) CX3CR1BII036 30mg/kg 17 6.8 52 (p < 0.01) (2×/week)

Both CX3CR1BII00313 and CX3CR1BII036 significantly inhibited plaqueprogression when dosed twice weekly. This correlated with coverage asplasma levels of these molecules could be confirmed to be maintainedthroughout the study. For once weekly dosing of CX3CR1BII00313,detectable plasma levels were not maintained and this correlated withthe lack of significant efficacy observed after 6 weeks of treatment.Neither molecule significantly affected plasma cholesterol ortriglyceride levels.

Example 14 Nanobody Ninding to Primary Human and Cynomolgus Monkey CD14+Cells in Whole Blood

Competition FACS with Formatted Sequence Optimized Anti-CX3CR1 Nanobody

To confirm binding of the formatted sequence optimized anti-CX3CR1Nanobody to human primary cells, CX3CR1BII00313 was demonstrated tocompete for the binding of A647 labeled CX3CR1BII018 (A647-018) to CD14+cells in a competition FACS assay in whole blood. Briefly, a mouseanti-human CD14 antibody conjugated with eFluor 450 (eBioscience, SanDiego, Calif., USA) was diluted 1:10 in EDTA treated whole blood from ahealthy human donor. 40 μl well was added to 96 well polystyrene roundbottom plate followed by 10 μl well of CX3CR1BII00313 diluted in StainBuffer with BSA (BD Pharmingen) at a final concentration ranging from100 nM to 0.002 pM and the samples were incubated for 20 minutes at roomtemperature. 10 μl/well of A647-018 in Stain Buffer was then added toyield a final concentration of 1 nM (the EC80 of A647-018 binding) andthe samples were incubated for an additional 20 minutes at roomtemperature. 220 μl/well of 1-Step Fix/Lyse solution (eBioscience) wasthen added. After a 10 minute room temperature incubation the cells werepelleted, washed twice in Stain buffer and resuspended in this buffer.The samples were analyzed on a BD LSR II flow cytometer. The medianfluorescence intensity for AlexaFluor 647 was quantified for the gateCD14 positive cell population. CX3CR1BII00313 potently inhibited thebinding of A647-018 to CD14 positive cells in human blood with an 1050of 0.35 nM (n=8).

To confirm binding of the formatted sequence optimized anti-CX3CR1Nanobody to cynomolgus monkey primary cells, CX3CR1BII00313 wasdemonstrated to compete for the binding of A647 labeled CX3CR1BII018(A647-018) to CD14+ cells in a competition FACS assay in cynomolgusmonkey whole blood. The method used was analogous to that outlined aboveexcept the final concentration of A647-018 was 3 nM (the EC80 ofA647-018 binding) and ACK lysing buffer (Life Technologies) was usedinstead of the 1-Step Fix/Lyse solution. The cells were resuspended inStain buffer supplemented with 1% formaldehyde prior to analysis. CX3CR1BII00313 potently inhibited the binding of A647-018 to CD14 positivecells in cynomolgus monkey blood with an IC50 of 0.43 nM (n=4).

Example 15 Pharmacokinetics (PK) in Cynomolgus Monkeys

A pharmacokinetic study was conducted in naïve male cynomolgus monkeys(Macaca fascicularis) 2-5 years of age with a body weight range between2.4-3.5 kg. The monkeys were divided into four treatment groups. Group 1(n=3) received 0.2 mg/kg of CX3CR1 BII00313 i.v.; Group 2 (n=3) received2 mg/kg of CX3CR1BII00313 i.v.; Group 3 (n=3) received 2 mg/kgCX3CR1BII00313 s.c. and Group 4 (n=3) received 5 mg/kg CX3CR1 BII00313i.v. CX3CR1BII00313 was administered as a 2 mg/ml solution in citratebuffer (20 mM sodium citrate/115 mM sodium chloride, pH 6.0). Bloodsamples were collected over 6 weeks from a peripheral vein into serumseparator tubes for PK analysis.

Serum samples were analyzed using a MSD (Meso Scale Discovery) format.Briefly, a biotinylated anti-Nanobody antibody was bound to a MSDstandard streptavidin plate (Meso Scale Discovery, Rockville, Md., USA).The plates were washed with 0.05% Tween 20 in phosphate buffered salineand blocked with 5% w/v of SeraCare BSA (SeraCare Life Sciences,Milford, Mass., USA) prior to incubation with serum samples. CX3CR1BII00313 was detected utilizing a sulfo-labeled anti-Nanobody Nanobodyand the plates were analyzed on a Sector Imager 2400 (Meso ScaleDiscovery). Varying concentrations of CX3CR1 BII0313 from 5000 to 0.5ng/ml in 5% monkey serum were used as standards. Target engagement wasassessed by monitoring levels of free CX3CR1 on CD14+ gated monocytes.This assay was analogous to the competition FACS assay summarized inExample 14 except no additional CX3CR1 BII00313 was added. Serum sampleswere also monitored for the presence of primate anti-human antibodies(PAHA) as they may impact assessment of PK and free CX3CR1.

ForteBio RED96 was used for detection of PAHA. Briefly, biotinylatedCX3CR1BII0313 was captured over streptavidin sensors. Pooled naïvemonkey serum was then used as a negative control to calculate cut-offvalue (defined as two fold above the average binding signal of naïvesera). All serum samples were diluted 20 fold in buffer and the PAHAresponse was determined to be positive if the binding signal was greaterthan the cut-off value.

Data for time points following detection of PAHA were excluded fromPK/PD analysis. The PK data are summarized in Table 44 below.

TABLE 44 Pharmacokinetic parameters of CX3CR1BII00313 Dose normalized CLT_(1/2) MRT AUC(0-14 d) Dose (mg/kg) (mL/day/kg) (day) (day) (nM · d) F% IV 0.2 113 1 1 ** IV 2.0 9 ± 1 9 ± 2 8 ± 2 56530 IV 5.0 ** ** ** 58604SC 2.0 54 ** insufficient data for characterization of terminal phase

Clearance and half-life at 2.0 mg/kg i.v. were 9.4 mL/d/kg and 9.6 days,respectively. At 0.2 mg/kg i.v., clearance was substantially higher (113mL/d/kg) consistent with saturable target-mediated disposition (TMD)pharmacokinetics. Dose-adjusted AUC_((0-14d)) was comparable between the2 and 5 mg/kg i.v. doses suggesting saturation of TMD at the 2 mg/kgdose. Exposure at 2 weeks following either i.v. or s.c. Nanobodyadministration was >70 nM and bioavailability after s.c. administrationwas 54%. Free receptor tracked with exposure with greater than 90%target coverage maintained at exposures >10 nM.

The invention claimed is:
 1. A polypeptide comprising an anti-CX3CR1(anti-CX3 chemokine receptor 1) immunoglobulin single variable domain,wherein said polypeptide comprises a CDR1, CDR2 and CDR3 having theamino acid sequences set forth in SEQ ID No: 141, 162 and 197,respectively; or SEQ ID No: 141, 163 and 197, respectively; or SEQ IDNo: 141, 164 and 197, respectively; or SEQ ID No: 141, 166 and 197,respectively; or SEQ ID No: 141, 167 and 197, respectively; or SEQ IDNo: 141, 168 and 197, respectively; or SEQ ID No: 141, 169 and 197,respectively; or SEQ ID No: 141, 170 and 197, respectively; or SEQ IDNo: 141, 171 and 197, respectively; or SEQ ID No: 141, 174 and 197,respectively; or SEQ ID No: 141, 175 and 197, respectively; or SEQ IDNo: 141, 214 and 197, respectively; or SEQ ID No: 141, 215 and 197,respectively; or SEQ ID No: 141, 216 and 197, respectively; or SEQ IDNo: 141, 217 and 197, respectively; or SEQ ID No: 141, 218 and 197,respectively; or SEQ ID No: 141, 219 and 197, respectively; or SEQ IDNo: 141, 220 and 197, respectively; or SEQ ID No: 142, 165 and 197,respectively; or SEQ ID No: 142, 173 and 197, respectively; or SEQ IDNo: 143, 164 and 197, respectively; or SEQ ID No: 144, 172 and 197,respectively; or SEQ ID No: 145, 172 and 197, respectively; or SEQ IDNo: 213, 221 and 197, respectively; or SEQ ID No: 213, 214 and 197,respectively; wherein said polypeptide is capable of blocking thebinding of human fractalkine to human CX3CR1.
 2. The polypeptideaccording to claim 1, wherein said anti-CX3CR1 immunoglobulin singlevariable domain consists essentially of four framework regions (FR1,FR2, FR3 and FR4) and three complementary determining regions (CDR1,CDR2 and CDR3).
 3. The polypeptide according to claim 1, wherein saidanti-CX3CR1 immunoglobulin single variable domain is a VH, VL, VHH,camelized VH, or VHH that is optimized for stability, potency,manufacturability and/or similarity to human framework regions.
 4. Thepolypeptide according to claim 1, wherein said polypeptide has anaffinity to human CX3CR1 at: c) an EC50 of less than or equal to 10 nMas determined by cell binding FACS (fluorescence activated cellsorting); or d) an IC50 of less than or equal to 10 nM as determined bycompetition FACS.
 5. The polypeptide according to claim 1, wherein saidpolypeptide blocks the binding of human fractalkine to human CX3CR1 atan IC50 of less than or equal to 300 nM.
 6. The polypeptide according toclaim 1, wherein said CDR3 has the amino acid sequence ofAsp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyr (SEQ ID NO: 186).
 7. Thepolypeptide according to claim 1, wherein said polypeptide comprises aCDR1, CDR2 and CDR3 having the amino acid sequences set forth in: SEQ IDNo: 147, 178 and 192, respectively; or SEQ ID No: 147, 179 and 192,respectively; or SEQ ID No: 147, 179 and 194, respectively; or SEQ IDNo: 148, 179 and 193, respectively; or SEQ ID No: 149, 179 and 192,respectively; or SEQ ID No: 149, 180 and 192, respectively; or SEQ IDNo: 149, 181 and 192, respectively; or SEQ ID No: 149, 183 and 192,respectively; or SEQ ID No: 149, 185 and 192, respectively; or SEQ IDNo: 150, 179 and 194, respectively; or SEQ ID No: 150, 182 and 194,respectively; or SEQ ID No: 151, 179 and 193, respectively; or SEQ IDNo: 151, 182 and 194, respectively; or SEQ ID No: 151, 184 and 196,respectively; or SEQ ID No: 152, 179 and 195, respectively; or SEQ IDNo: 153, 179 and 194, respectively; or SEQ ID No: 154, 182 and 194,respectively; or SEQ ID No: 155, 179 and 195, respectively; or SEQ IDNo: 156, 181 and 192, respectively; or SEQ ID No: 157, 179 and 194,respectively; or SEQ ID No: 158, 179 and 192, respectively; or SEQ IDNo: 159, 178 and 192, respectively; or SEQ ID No: 160, 179 and 194,respectively; or SEQ ID No: 161, 179 and 194, respectively.
 8. Thepolypeptide according to claim 1, wherein the polypeptide comprises aCDR1, CDR2 and CDR3 having amino acid sequences set forth in: SEQ IDNO's: 141, 164 and 186, respectively, SEQ ID NO's: 141, 162 and 186,respectively, SEQ ID NO's: 213, 214 and 186 respectively, or SEQ IDNO's: 213, 221 and 186 respectively.
 9. The polypeptide according toclaim 1, wherein said anti-CX3CR1 immunoglobulin single variable domainis a VHH domain comprising the sequence set forth in: e) the amino acidsequence of SEQ ID NO: 3; f) an amino acid sequence that has at least90% amino acid identity with the amino acid sequences of SEQ ID NO: 3;or g) an amino acid sequence of any one of SEQ ID NO: 1-48, 121-140 or222-224.
 10. The polypeptide according to claim 1, wherein saidanti-CX3CR1 immunoglobulin single variable domain is a VHH domaincomprising the sequence set forth in: e) the amino acid sequence of SEQID NO: 67; f) an amino acid sequence that has at least 90% amino acididentity with the amino acid sequences of SEQ ID NO: 67; or g) an aminoacid sequence of any one of SEQ ID NO: 53-120.
 11. The polypeptideaccording to claim 1, wherein said anti-CX3CR1 immunoglobulin singlevariable domain comprises the sequence set forth in SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 121-140 or SEQ ID NO: 222-224.
 12. The polypeptideaccording to claim 1, wherein said immunoglobulin single variable domaineither a) cross-blocks the binding of, or b) is cross-blocked frombinding by at least one of the immunoglobulin single variable domains ofSEQ ID NO's: 1-120, 121-140 and 222-224 to CX3CR1.
 13. The polypeptideaccording to claim 1, wherein the polypeptide further comprises ahalf-life extending moiety.
 14. The polypeptide according to claim 13,wherein said half-life extending moiety is covalently linked to saidpolypeptide and is selected from the group consisting of an albuminbinding moiety, such as an anti-albumin immunoglobulin domain, atransferrin binding moiety, such as an anti-transferrin immunoglobulindomain, a polyethylene glycol molecule, a recombinant polyethyleneglycol molecule, human serum albumin, a fragment of human serum albumin,an albumin binding peptide or a Fc domain.
 15. The polypeptide accordingto claim 13, wherein said half-life extending moiety consists of ananti-albumin immunoglobulin single variable domain.
 16. The polypeptideaccording to claim 15, wherein the immunoglobulin single variable domainis selected from a VHH domain, a humanized VHH domain, a camelized VHdomain, a domain antibody, a single domain antibody and/or “dAb”s.
 17. Apolypeptide comprising an anti-CX3CR1 (anti-CX3 chemokine receptor 1)immunoglobulin single variable domain 1, wherein said polypeptidecomprises a CDR1, CDR2 and CDR3 having the amino acid sequences setforth in: SEQ ID No: 146, 176 and 191, respectively; or SEQ ID No: 146,177 and 191, respectively.
 18. The polypeptide according to claim 17,wherein said anti-CX3CR1 immunoglobulin single variable domain is a VHHdomain comprising the sequence set forth in: e) the amino acid sequenceof SEQ ID NO: 49; f) an amino acid sequence that has at least 95% aminoacid identity with the amino acid sequences of SEQ ID NO: 49; or g) anamino acid sequence of any one of SEQ ID NO: 49-52.